EP4275686A1 - Combination therapy for cancer comprising a cdc7 inhibitor and a cdk8 inihibitor or a ccnc inhibitor - Google Patents
Combination therapy for cancer comprising a cdc7 inhibitor and a cdk8 inihibitor or a ccnc inhibitor Download PDFInfo
- Publication number
- EP4275686A1 EP4275686A1 EP22172868.6A EP22172868A EP4275686A1 EP 4275686 A1 EP4275686 A1 EP 4275686A1 EP 22172868 A EP22172868 A EP 22172868A EP 4275686 A1 EP4275686 A1 EP 4275686A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cdk8
- ccnc
- inhibitor
- cdc7
- cdc7i
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003112 inhibitor Substances 0.000 title claims abstract description 168
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 91
- 201000011510 cancer Diseases 0.000 title claims abstract description 63
- 238000002648 combination therapy Methods 0.000 title claims abstract description 10
- 101150065030 cdc7 gene Proteins 0.000 title description 2
- 101150049479 CCNC gene Proteins 0.000 title 1
- 101150090188 Cdk8 gene Proteins 0.000 title 1
- 101150112625 SSN3 gene Proteins 0.000 title 1
- 101100150415 Schizosaccharomyces pombe (strain 972 / ATCC 24843) srb10 gene Proteins 0.000 title 1
- 101000945740 Homo sapiens Cell division cycle 7-related protein kinase Proteins 0.000 claims abstract description 155
- 102100034744 Cell division cycle 7-related protein kinase Human genes 0.000 claims abstract description 154
- 102000002428 Cyclin C Human genes 0.000 claims abstract description 112
- 108010068155 Cyclin C Proteins 0.000 claims abstract description 112
- 229940123484 Cyclin-dependent kinase 8 inhibitor Drugs 0.000 claims abstract description 41
- 238000011282 treatment Methods 0.000 claims abstract description 35
- 230000002265 prevention Effects 0.000 claims abstract description 12
- 229940043355 kinase inhibitor Drugs 0.000 claims abstract description 11
- 239000003757 phosphotransferase inhibitor Substances 0.000 claims abstract description 11
- 102100024456 Cyclin-dependent kinase 8 Human genes 0.000 claims description 231
- 230000014509 gene expression Effects 0.000 claims description 44
- JJWLXRKVUJDJKG-VIFPVBQESA-N XL413 Chemical compound C12=CC(Cl)=CC=C2OC(C(N2)=O)=C1N=C2[C@@H]1CCCN1 JJWLXRKVUJDJKG-VIFPVBQESA-N 0.000 claims description 42
- 238000000034 method Methods 0.000 claims description 33
- 239000000203 mixture Substances 0.000 claims description 23
- 230000002401 inhibitory effect Effects 0.000 claims description 18
- XGVXKJKTISMIOW-ZDUSSCGKSA-N simurosertib Chemical compound N1N=CC(C=2SC=3C(=O)NC(=NC=3C=2)[C@H]2N3CCC(CC3)C2)=C1C XGVXKJKTISMIOW-ZDUSSCGKSA-N 0.000 claims description 16
- 101000980937 Homo sapiens Cyclin-dependent kinase 8 Proteins 0.000 claims description 15
- 230000003247 decreasing effect Effects 0.000 claims description 15
- 101000980770 Homo sapiens Cyclin-C Proteins 0.000 claims description 13
- -1 RVU120 Chemical compound 0.000 claims description 13
- 238000002560 therapeutic procedure Methods 0.000 claims description 13
- 230000002950 deficient Effects 0.000 claims description 11
- 206010012818 diffuse large B-cell lymphoma Diseases 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- VNADJTWHOAMTLY-UHFFFAOYSA-N 4-[2-[6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl]ethylamino]quinazoline-6-carbonitrile Chemical compound C1CN(C)CCN1C(=O)C1=CC=C(C=C(CCNC=2C3=CC(=CC=C3N=CN=2)C#N)C=C2)C2=C1 VNADJTWHOAMTLY-UHFFFAOYSA-N 0.000 claims description 7
- 208000031671 Large B-Cell Diffuse Lymphoma Diseases 0.000 claims description 7
- 230000009467 reduction Effects 0.000 claims description 7
- DKXHSOUZPMHNIZ-UHFFFAOYSA-N 2-pyridin-4-yl-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridin-4-one Chemical compound C=1C=2C(=O)NCCC=2NC=1C1=CC=NC=C1 DKXHSOUZPMHNIZ-UHFFFAOYSA-N 0.000 claims description 6
- ODRITQGYYWHQGM-INIZCTEOSA-N ClC1=CC=C(C=C1)[C@H]1N(CCC1)C(=O)C=1C=C2C(=NC=1)NN=C2C Chemical compound ClC1=CC=C(C=C1)[C@H]1N(CCC1)C(=O)C=1C=C2C(=NC=1)NN=C2C ODRITQGYYWHQGM-INIZCTEOSA-N 0.000 claims description 6
- VPJXPDLMACOGIZ-UHFFFAOYSA-N 8-[2-amino-3-chloro-5-(1-methylindazol-5-yl)pyridin-4-yl]-2,8-diazaspiro[4.5]decan-1-one Chemical compound C=1C=C2N(C)N=CC2=CC=1C1=CN=C(N)C(Cl)=C1N(CC1)CCC21CCNC2=O VPJXPDLMACOGIZ-UHFFFAOYSA-N 0.000 claims description 5
- 206010039491 Sarcoma Diseases 0.000 claims description 5
- HFBNZZVVRMLDJO-JLHYYAGUSA-N ethyl 4-hydroxy-5-[(Z)-pyrrolo[2,3-b]pyridin-3-ylidenemethyl]-2-[[4-(2,2,2-trifluoroethyl)piperazin-1-yl]amino]furan-3-carboxylate Chemical compound CCOC(=O)c1c(O)c(\C=C2/C=Nc3ncccc23)oc1NN1CCN(CC(F)(F)F)CC1 HFBNZZVVRMLDJO-JLHYYAGUSA-N 0.000 claims description 5
- AFUHWZUYZJGVKM-UHFFFAOYSA-N 4-[2-[6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl]ethylamino]quinoline-6-carbonitrile Chemical compound CN1CCN(CC1)C(=O)C=1C=C2C=CC(=CC2=CC=1)CCNC1=CC=NC2=CC=C(C=C12)C#N AFUHWZUYZJGVKM-UHFFFAOYSA-N 0.000 claims description 4
- LBFYQISQYCGDDW-UHFFFAOYSA-N CCT251545 Chemical compound C1=NN(C)C=C1C1=CC=C(C=2C(=C(Cl)C=NC=2)N2CCC3(C(NCC3)=O)CC2)C=C1 LBFYQISQYCGDDW-UHFFFAOYSA-N 0.000 claims description 4
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims description 4
- 210000004185 liver Anatomy 0.000 claims description 4
- XWPCYYOZOJKYKQ-UHFFFAOYSA-N 1-(2-chloroethyl)-3-[2-[2-[[2-chloroethyl(nitroso)carbamoyl]amino]ethyldisulfanyl]ethyl]-1-nitrosourea Chemical compound ClCCN(N=O)C(=O)NCCSSCCNC(=O)N(N=O)CCCl XWPCYYOZOJKYKQ-UHFFFAOYSA-N 0.000 claims description 3
- QSYBDNXNOJIKML-UHFFFAOYSA-N 2-amino-6-methyl-4-(3-pyridinyl)-5,6,7,8-tetrahydroquinoline-3-carbonitrile Chemical compound C=12CC(C)CCC2=NC(N)=C(C#N)C=1C1=CC=CN=C1 QSYBDNXNOJIKML-UHFFFAOYSA-N 0.000 claims description 3
- XBJCNHGQFJFCOY-UHFFFAOYSA-N 4-(2-phenylethylamino)quinazoline-6-carbonitrile Chemical compound C12=CC(C#N)=CC=C2N=CN=C1NCCC1=CC=CC=C1 XBJCNHGQFJFCOY-UHFFFAOYSA-N 0.000 claims description 3
- LHUAALWRORLTCJ-UHFFFAOYSA-N 4-[1-(2-methyl-3H-benzimidazol-5-yl)imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine dihydrochloride Chemical compound Cl.Cl.Cc1nc2ccc(cc2[nH]1)-n1c(nc2cnccc12)-c1nonc1N LHUAALWRORLTCJ-UHFFFAOYSA-N 0.000 claims description 3
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims description 3
- YLJSAFSDGFNKAC-UHFFFAOYSA-N CN1C2=C(C=C(C=C2)C3=C(C4=C(NN=C4N=C3)Cl)N5CCC6(CCNC6=O)CC5)C=N1 Chemical compound CN1C2=C(C=C(C=C2)C3=C(C4=C(NN=C4N=C3)Cl)N5CCC6(CCNC6=O)CC5)C=N1 YLJSAFSDGFNKAC-UHFFFAOYSA-N 0.000 claims description 3
- JECHBTRAPARMGI-GFTKVEOVSA-N C[C@]12CC[C@@H](C[C@@H]1CC[C@@H]3[C@@H]2CC[C@]4([C@H]3CC[C@@H]4C5=CC6=C(C=C5)C=CN=C6)C)N(C)C(=O)CCOCCOCCOCCOCCNC7=CC=CC8=C7C(=O)N(C8=O)C9CCC(=O)NC9=O Chemical compound C[C@]12CC[C@@H](C[C@@H]1CC[C@@H]3[C@@H]2CC[C@]4([C@H]3CC[C@@H]4C5=CC6=C(C=C5)C=CN=C6)C)N(C)C(=O)CCOCCOCCOCCOCCNC7=CC=CC8=C7C(=O)N(C8=O)C9CCC(=O)NC9=O JECHBTRAPARMGI-GFTKVEOVSA-N 0.000 claims description 3
- 201000005969 Uveal melanoma Diseases 0.000 claims description 3
- 239000000090 biomarker Substances 0.000 claims description 3
- LTNZEXKYNRNOGT-UHFFFAOYSA-N dequalinium chloride Chemical compound [Cl-].[Cl-].C1=CC=C2[N+](CCCCCCCCCC[N+]3=C4C=CC=CC4=C(N)C=C3C)=C(C)C=C(N)C2=C1 LTNZEXKYNRNOGT-UHFFFAOYSA-N 0.000 claims description 3
- 229960001378 dequalinium chloride Drugs 0.000 claims description 3
- 201000006585 gastric adenocarcinoma Diseases 0.000 claims description 3
- 231100000844 hepatocellular carcinoma Toxicity 0.000 claims description 3
- 201000005825 prostate adenocarcinoma Diseases 0.000 claims description 3
- 206010005084 bladder transitional cell carcinoma Diseases 0.000 claims description 2
- 201000001528 bladder urothelial carcinoma Diseases 0.000 claims description 2
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 2
- 102100024170 Cyclin-C Human genes 0.000 claims 12
- 108010025415 Cyclin-Dependent Kinase 8 Proteins 0.000 description 217
- 210000004027 cell Anatomy 0.000 description 127
- 241000282414 Homo sapiens Species 0.000 description 63
- 108090000623 proteins and genes Proteins 0.000 description 62
- 150000001875 compounds Chemical class 0.000 description 57
- 230000005764 inhibitory process Effects 0.000 description 43
- 102100033145 Cyclin-dependent kinase 19 Human genes 0.000 description 40
- 101000944345 Homo sapiens Cyclin-dependent kinase 19 Proteins 0.000 description 35
- 108091027544 Subgenomic mRNA Proteins 0.000 description 34
- 108091033409 CRISPR Proteins 0.000 description 33
- 230000000694 effects Effects 0.000 description 31
- 150000003839 salts Chemical class 0.000 description 26
- 230000006820 DNA synthesis Effects 0.000 description 23
- 230000018199 S phase Effects 0.000 description 21
- 108020004459 Small interfering RNA Proteins 0.000 description 21
- 238000010354 CRISPR gene editing Methods 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 19
- 230000006870 function Effects 0.000 description 17
- 230000004543 DNA replication Effects 0.000 description 15
- 230000035755 proliferation Effects 0.000 description 15
- 238000013518 transcription Methods 0.000 description 15
- 230000035897 transcription Effects 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 13
- 230000007423 decrease Effects 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 230000022131 cell cycle Effects 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- 102000000490 Mediator Complex Human genes 0.000 description 11
- 108010080991 Mediator Complex Proteins 0.000 description 11
- 102100021070 Mediator of RNA polymerase II transcription subunit 12 Human genes 0.000 description 11
- 230000001028 anti-proliverative effect Effects 0.000 description 11
- 230000004663 cell proliferation Effects 0.000 description 11
- 239000000306 component Substances 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 101000614988 Homo sapiens Mediator of RNA polymerase II transcription subunit 12 Proteins 0.000 description 10
- 239000002253 acid Substances 0.000 description 10
- 239000012453 solvate Substances 0.000 description 10
- 230000000153 supplemental effect Effects 0.000 description 10
- 230000001225 therapeutic effect Effects 0.000 description 10
- 102000009572 RNA Polymerase II Human genes 0.000 description 9
- 108010009460 RNA Polymerase II Proteins 0.000 description 9
- 238000009826 distribution Methods 0.000 description 9
- 238000007481 next generation sequencing Methods 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 102100026161 Mediator of RNA polymerase II transcription subunit 13 Human genes 0.000 description 8
- 150000001204 N-oxides Chemical class 0.000 description 8
- 230000010076 replication Effects 0.000 description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 101001055427 Homo sapiens Mediator of RNA polymerase II transcription subunit 13 Proteins 0.000 description 7
- 125000003118 aryl group Chemical group 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000007812 deficiency Effects 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 150000002148 esters Chemical class 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 230000003287 optical effect Effects 0.000 description 7
- 230000003389 potentiating effect Effects 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 7
- 125000001424 substituent group Chemical group 0.000 description 7
- 230000002103 transcriptional effect Effects 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 101001028025 Homo sapiens Mdm2-binding protein Proteins 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 102100037572 Mdm2-binding protein Human genes 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 125000003342 alkenyl group Chemical group 0.000 description 6
- 125000000217 alkyl group Chemical group 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 239000002246 antineoplastic agent Substances 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 229910052794 bromium Inorganic materials 0.000 description 5
- 229910052801 chlorine Inorganic materials 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 229910052731 fluorine Inorganic materials 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 229940002612 prodrug Drugs 0.000 description 5
- 239000000651 prodrug Substances 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000010361 transduction Methods 0.000 description 5
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 4
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 4
- 101710179322 Cyclin-dependent kinase 19 Proteins 0.000 description 4
- 102100024457 Cyclin-dependent kinase 9 Human genes 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 101000980930 Homo sapiens Cyclin-dependent kinase 9 Proteins 0.000 description 4
- 229930182555 Penicillin Natural products 0.000 description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 4
- 108091000080 Phosphotransferase Proteins 0.000 description 4
- 102100037806 Telomere-associated protein RIF1 Human genes 0.000 description 4
- 125000000304 alkynyl group Chemical group 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 125000004429 atom Chemical group 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 230000003081 coactivator Effects 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 125000000623 heterocyclic group Chemical group 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 238000010348 incorporation Methods 0.000 description 4
- 229910052740 iodine Inorganic materials 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 125000004433 nitrogen atom Chemical group N* 0.000 description 4
- 229940049954 penicillin Drugs 0.000 description 4
- 102000020233 phosphotransferase Human genes 0.000 description 4
- 230000007115 recruitment Effects 0.000 description 4
- 238000009097 single-agent therapy Methods 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 229940124597 therapeutic agent Drugs 0.000 description 4
- 230000026683 transduction Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- GQXLWUCQESKBSC-UHFFFAOYSA-N 6,7-dibromo-5-methyl-2-piperazin-1-yl-1,3-diazatricyclo[6.3.1.04,12]dodeca-2,4,6,8(12)-tetraene hydrochloride Chemical compound Cl.N=1C(=C23)C(C)=C(Br)C(Br)=C2CCCN3C=1N1CCNCC1 GQXLWUCQESKBSC-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- 108091007914 CDKs Proteins 0.000 description 3
- 201000009030 Carcinoma Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101001095487 Homo sapiens Telomere-associated protein RIF1 Proteins 0.000 description 3
- 239000004721 Polyphenylene oxide Substances 0.000 description 3
- 108091027967 Small hairpin RNA Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- 125000004423 acyloxy group Chemical group 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 125000004432 carbon atom Chemical group C* 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000010304 firing Methods 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
- 125000005843 halogen group Chemical group 0.000 description 3
- 201000005787 hematologic cancer Diseases 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 230000011278 mitosis Effects 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 3
- 229920000570 polyether Polymers 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000004055 small Interfering RNA Substances 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 125000003944 tolyl group Chemical group 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 125000005023 xylyl group Chemical group 0.000 description 3
- 125000006701 (C1-C7) alkyl group Chemical group 0.000 description 2
- HQVZOORKDNCGCK-UHFFFAOYSA-N 2-[(2,4-dichlorophenyl)methyl]-4-(2,4,4-trimethylpentan-2-yl)phenol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(O)C(CC=2C(=CC(Cl)=CC=2)Cl)=C1 HQVZOORKDNCGCK-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 108010077544 Chromatin Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108050006400 Cyclin Proteins 0.000 description 2
- 102000016736 Cyclin Human genes 0.000 description 2
- 102000003903 Cyclin-dependent kinases Human genes 0.000 description 2
- 108090000266 Cyclin-dependent kinases Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108700039887 Essential Genes Proteins 0.000 description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 2
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000998810 Homo sapiens Insulin-like peptide INSL6 Proteins 0.000 description 2
- 101000619643 Homo sapiens Ligand-dependent nuclear receptor-interacting factor 1 Proteins 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010029260 Neuroblastoma Diseases 0.000 description 2
- 102000007399 Nuclear hormone receptor Human genes 0.000 description 2
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 229940124639 Selective inhibitor Drugs 0.000 description 2
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 208000037844 advanced solid tumor Diseases 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 125000000129 anionic group Chemical group 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 210000003483 chromatin Anatomy 0.000 description 2
- 229960003769 clofoctol Drugs 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 238000003235 crystal violet staining Methods 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 125000004185 ester group Chemical group 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 102000034356 gene-regulatory proteins Human genes 0.000 description 2
- 108091006104 gene-regulatory proteins Proteins 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 2
- 125000001072 heteroaryl group Chemical group 0.000 description 2
- 125000001183 hydrocarbyl group Chemical group 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 125000002883 imidazolyl group Chemical group 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 125000001041 indolyl group Chemical group 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 208000037843 metastatic solid tumor Diseases 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 2
- 125000001624 naphthyl group Chemical group 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 230000006780 non-homologous end joining Effects 0.000 description 2
- 239000000101 novel biomarker Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 201000008968 osteosarcoma Diseases 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 150000003003 phosphines Chemical class 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 150000003141 primary amines Chemical class 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 210000002307 prostate Anatomy 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 125000003373 pyrazinyl group Chemical group 0.000 description 2
- 125000004076 pyridyl group Chemical group 0.000 description 2
- 125000000714 pyrimidinyl group Chemical group 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 150000003335 secondary amines Chemical class 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000000528 statistical test Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000008093 supporting effect Effects 0.000 description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 150000003512 tertiary amines Chemical class 0.000 description 2
- 125000000335 thiazolyl group Chemical group 0.000 description 2
- 125000001544 thienyl group Chemical group 0.000 description 2
- 239000010936 titanium Substances 0.000 description 2
- 229910052719 titanium Inorganic materials 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- UDQTXCHQKHIQMH-KYGLGHNPSA-N (3ar,5s,6s,7r,7ar)-5-(difluoromethyl)-2-(ethylamino)-5,6,7,7a-tetrahydro-3ah-pyrano[3,2-d][1,3]thiazole-6,7-diol Chemical compound S1C(NCC)=N[C@H]2[C@@H]1O[C@H](C(F)F)[C@@H](O)[C@@H]2O UDQTXCHQKHIQMH-KYGLGHNPSA-N 0.000 description 1
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- 125000004209 (C1-C8) alkyl group Chemical group 0.000 description 1
- 125000006656 (C2-C4) alkenyl group Chemical group 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- NHQDETIJWKXCTC-UHFFFAOYSA-N 3-chloroperbenzoic acid Chemical compound OOC(=O)C1=CC=CC(Cl)=C1 NHQDETIJWKXCTC-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- 125000004648 C2-C8 alkenyl group Chemical group 0.000 description 1
- 102000042858 CMGC Ser/Thr protein kinase family Human genes 0.000 description 1
- 108091082290 CMGC Ser/Thr protein kinase family Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 1
- 208000010667 Carcinoma of liver and intrahepatic biliary tract Diseases 0.000 description 1
- 208000003163 Cavernous Hemangioma Diseases 0.000 description 1
- 229940084129 Cdc7 kinase inhibitor Drugs 0.000 description 1
- 102000009016 Cholera Toxin Human genes 0.000 description 1
- 108010049048 Cholera Toxin Proteins 0.000 description 1
- 208000006332 Choriocarcinoma Diseases 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- 101710179261 Cyclin-dependent kinase 11 Proteins 0.000 description 1
- 101710122917 Cyclin-dependent kinase 11B Proteins 0.000 description 1
- 102100033144 Cyclin-dependent kinase 18 Human genes 0.000 description 1
- 102100026810 Cyclin-dependent kinase 7 Human genes 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 239000012623 DNA damaging agent Substances 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical compound C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 102100036935 Ewing's tumor-associated antigen 1 Human genes 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 108010008945 General Transcription Factors Proteins 0.000 description 1
- 102000006580 General Transcription Factors Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010073069 Hepatic cancer Diseases 0.000 description 1
- 101000911952 Homo sapiens Cyclin-dependent kinase 7 Proteins 0.000 description 1
- 101000851494 Homo sapiens Ewing's tumor-associated antigen 1 Proteins 0.000 description 1
- 101001056180 Homo sapiens Induced myeloid leukemia cell differentiation protein Mcl-1 Proteins 0.000 description 1
- 101001017597 Homo sapiens Mediator of RNA polymerase II transcription subunit 12-like protein Proteins 0.000 description 1
- 101001017592 Homo sapiens Mediator of RNA polymerase II transcription subunit 13-like Proteins 0.000 description 1
- 101000721172 Homo sapiens Protein DBF4 homolog A Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102100026539 Induced myeloid leukemia cell differentiation protein Mcl-1 Human genes 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 229930194542 Keto Natural products 0.000 description 1
- 125000002842 L-seryl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])O[H] 0.000 description 1
- 208000018142 Leiomyosarcoma Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 101710179284 Mediator of RNA polymerase II transcription subunit 12 Proteins 0.000 description 1
- 102100034160 Mediator of RNA polymerase II transcription subunit 12-like protein Human genes 0.000 description 1
- 101710179416 Mediator of RNA polymerase II transcription subunit 13 Proteins 0.000 description 1
- 102100034164 Mediator of RNA polymerase II transcription subunit 13-like Human genes 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 102100030979 Methylmalonyl-CoA mutase, mitochondrial Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 1
- 229920002274 Nalgene Polymers 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 208000035327 Oestrogen receptor positive breast cancer Diseases 0.000 description 1
- 201000010133 Oligodendroglioma Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 108010039798 PCTAIRE-3 protein kinase Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000005877 Peptide Initiation Factors Human genes 0.000 description 1
- 108010044843 Peptide Initiation Factors Proteins 0.000 description 1
- 206010048734 Phakomatosis Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 102100025198 Protein DBF4 homolog A Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric Acid Chemical class [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 101710103331 Telomere-associated protein RIF1 Proteins 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 102000000504 Tumor Suppressor p53-Binding Protein 1 Human genes 0.000 description 1
- 108010041385 Tumor Suppressor p53-Binding Protein 1 Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 102000006668 UniProt protein families Human genes 0.000 description 1
- 108020004729 UniProt protein families Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000008383 Wilms tumor Diseases 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 125000003668 acetyloxy group Chemical group [H]C([H])([H])C(=O)O[*] 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229910001413 alkali metal ion Inorganic materials 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001409 amidines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000011394 anticancer treatment Methods 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- JXLHNMVSKXFWAO-UHFFFAOYSA-N azane;7-fluoro-2,1,3-benzoxadiazole-4-sulfonic acid Chemical compound N.OS(=O)(=O)C1=CC=C(F)C2=NON=C12 JXLHNMVSKXFWAO-UHFFFAOYSA-N 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000008236 biological pathway Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- SKOLWUPSYHWYAM-UHFFFAOYSA-N carbonodithioic O,S-acid Chemical compound SC(S)=O SKOLWUPSYHWYAM-UHFFFAOYSA-N 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 150000001733 carboxylic acid esters Chemical class 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 150000001767 cationic compounds Chemical class 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000008783 cell intrinsic mechanism Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 238000012650 click reaction Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 229940047120 colony stimulating factors Drugs 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 229940125936 compound 42 Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 239000001064 degrader Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 230000001909 effect on DNA Effects 0.000 description 1
- 150000002081 enamines Chemical class 0.000 description 1
- 201000003914 endometrial carcinoma Diseases 0.000 description 1
- 201000006828 endometrial hyperplasia Diseases 0.000 description 1
- 125000002587 enol group Chemical group 0.000 description 1
- 150000002085 enols Chemical class 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 201000005619 esophageal carcinoma Diseases 0.000 description 1
- 201000007281 estrogen-receptor positive breast cancer Diseases 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 150000002241 furanones Chemical class 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- GVVPGTZRZFNKDS-JXMROGBWSA-N geranyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O GVVPGTZRZFNKDS-JXMROGBWSA-N 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 201000002222 hemangioblastoma Diseases 0.000 description 1
- 201000011066 hemangioma Diseases 0.000 description 1
- 208000006359 hepatoblastoma Diseases 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000003331 infrared imaging Methods 0.000 description 1
- 238000011221 initial treatment Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229910001411 inorganic cation Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000005264 laryngeal carcinoma Diseases 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 201000002250 liver carcinoma Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229960003194 meglumine Drugs 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000011294 monotherapeutic Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- GTWJETSWSUWSEJ-UHFFFAOYSA-N n-benzylaniline Chemical compound C=1C=CC=CC=1CNC1=CC=CC=C1 GTWJETSWSUWSEJ-UHFFFAOYSA-N 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 208000007538 neurilemmoma Diseases 0.000 description 1
- 125000002560 nitrile group Chemical group 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 125000000018 nitroso group Chemical group N(=O)* 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 230000006508 oncogene activation Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 150000002892 organic cations Chemical class 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000009038 pharmacological inhibition Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 150000003140 primary amides Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 201000001514 prostate carcinoma Diseases 0.000 description 1
- 238000001273 protein sequence alignment Methods 0.000 description 1
- ORKUYZDMEWAUEZ-UHFFFAOYSA-N pyrrolo[3,2-b]pyridin-2-one Chemical class N1=CC=CC2=NC(=O)C=C21 ORKUYZDMEWAUEZ-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000014493 regulation of gene expression Effects 0.000 description 1
- 201000010174 renal carcinoma Diseases 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 230000028617 response to DNA damage stimulus Effects 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 206010039667 schwannoma Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003334 secondary amides Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 201000008261 skin carcinoma Diseases 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 235000019187 sodium-L-ascorbate Nutrition 0.000 description 1
- 239000011755 sodium-L-ascorbate Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000011044 succinic acid Nutrition 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229960000281 trometamol Drugs 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/472—Non-condensed isoquinolines, e.g. papaverine
- A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4739—Cyclin; Prad 1
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to a combination therapy to treat or prevent cancer, including methods of treatment or prevention, uses, compositions and kits for treating cancer in a subject.
- CDC7 kinase encoded by the CDC7 gene is considered to be a key switch for DNA replication and its depletion by siRNA causes cancer cells to stop DNA synthesis.
- CDC7i a number of CDC7 kinase inhibitors
- CDC7 inhibitors will be included as a component of chemotherapy with other agents.
- Previous studies have shown that inhibition of the ATR kinase can modify the cell sensitivity to CDC7i, however enhancement of CDC7i anti-cancer activity is observed only at very high doses of CDC7i while suppression is observed at lower doses (Guo et al., 2021; Iwai et al., 2019; Rainey et al., 2020).
- Such a combination therapy does not adequately address the clinical need.
- An aim of the present invention is to provide an alternative or improved therapy for cancer.
- a method of treatment or prevention of cancer in a subject comprising the administration of a Cell Division Cycle 7 kinase inhibitor (CDC7i) in combination with:
- the invention advantageously identifies CDK8 as a protein, that assists CDC7 in DNA replication. It is found that pharmacological inhibition of CDK8 has a negligible effect on DNA replication and cell division, but greatly enforces the anti-proliferative activity of CDC7 inhibitors when used in combination. Thus, the use of CDK8 inhibitors (and/or inhibitors of its cognate CCNC) in combination with CDC7 inhibitors greatly improve the efficacy of the latter anti-cancer drug. CDK8 inhibitors are being developed as potential anti-cancer agents with 2 reported clinical trials (ClinicalTrials.gov):
- the present invention finds for the first time that CDC7i and CDK8i (additionally or alternative CCNCi) synergise in stopping DNA replication and cell proliferation.
- combination therapy using CDC7i and CDK8i can be more efficacious and less toxic than CDC7 inhibitor monotherapy for the treatment of solid cancers, blood cancers and other proliferative diseases.
- the CDC7i may bind to CDC7.
- the binding to CDC7 is preferential or specific binding.
- the CDC7i may be a small molecule (i.e. less than 900Da).
- the CDC7i may be a small molecule ATP-competitive CDC7 inhibitor,
- the CDC7i may comprise or consist of XL413, or a functional derivative or variant thereof having CDC7 inhibition activity.
- the CDC7i may comprise or consist of any one of the group of CDC7 inhibitors selected from PHA-767491 (Montagnoli et al., 2008), XL413 (BMS-863233; Clinicaltrials.gov ID: NCT00838890 or NCT00886782) (Koltun et al., 2012), AS-0141 (Irie et al., 2021), Dequalinium Chloride, Clofoctol (Cheng et al., 2018), LY3143921 (Clinicaltrials.gov ID: NCT03096054), TQB3824 (Clinicaltrials.gov ID: NCT05028218), NMS-1116354 (Clinicaltrials.gov ID: NCT01016327 or NCT01092052) and TAK-931 (Clinicaltrials.gov ID: NCT02699749) (Iwai et al., 2019); or combinations thereof.
- PHA-767491 (PubChem CID: 11715767) has the formula:
- PHA-767491 Active variants of PHA-767491 can be found in Vanotti et al. (Vanotti et al., 2008), Menichincheri et al. (Menichincheri et al., 2009), Ermoli et al. (Ermoli et al., 2009), and Menichincheri et al. (Menichincheri et al., 2010), which are herein incorporated by reference.
- XL413 (BMS-863233, PubChem CID: 135564632) has the formula:
- Dequalinium chloride (DQC, PubChem CID: 2993) has the formula:
- Clofoctol (PubChem CID: 2799) has the formula:
- LY3143921 (PubChem CID: 137794949) has the formula:
- TQB3203 has the formula:
- NMS-1116354 has the formula:
- TAK-931 (Simurosurtib, PubChem CID: 136603526) has the formula:
- TAK-931 is described in Iwai K et al. (Iwai et al., 2019), which is herein incorporated by reference. Active variants of TAK-931 may be found in Kurasawa et al. (Kurasawa et al., 2020) and Kurasawa et al. (Kurasawa et al., 2017), both of which are herein incorporated by reference.
- the CDC7i comprises or consists of TAK931 or AS-0114. In one embodiment, the CDC7i comprises or consists of TAK931. In another embodiment, the CDC7i comprises or consists of AS-0114. In another preferred embodiment, the CDC7i comprises or consists of XL413 (BMS-863233).
- the CDC7i is a nucleic acid, such as an inhibitory RNA molecule.
- the CDC7i may be an siRNA capable of inhibiting the expression of CDC7, such as inhibiting the transcription of the gene encoding CDC7 or inhibiting translation of mRNA encoding CDC7.
- the CDC7i such as a siRNA, may bind the sequence encoding CDC7 or a complementary sequence thereof.
- the siRNA may be 20-30 nucleotides long.
- the siRNA may comprise or consist of RNA or may comprise or consist of nucleotide analogues.
- the CDC7i may specifically bind (i.e. hybridise with) a sequence comprising or consisting of SEQ ID NO: 1.
- the CDC7i may be any CDC7 siRNA described herein.
- the CDK8i may be a dual CDK8/CDK19 inhibitor.
- the CDK8i inhibitor may be specific for CDK8 and not CDK19.
- the CDK8i may not significantly inhibit CDK19.
- the CDK8i may comprise or consist of BI1347, or a functional derivative or variant thereof having CDK8 inhibition activity.
- the CDK8i may comprise or consist of any one of the group of CDK8 inhibitors selected from BI1347 (Hofmann et al., 2020), RVU120 (SEL120-34A, Clinicaltrials.gov ID: NCT04021368) (Johannessen et al., 2017), Senexin A (Porter et al., 2012), BCD-115 (Senexin B, Clinicaltrials.gov ID: NCT03065010) (Johannessen et al., 2017), Senexin C (Zhang et al., 2022), MSC2530818 (Czodrowski et al., 2016; Mallinger et al., 2016), AS2863619 (Akamatsu et al., 2019), 16-didehydro-cortistatin A (DCA) (Arnett et al., 2021), BRD6989 (Johannessen et al., 2017), CCT-251545 (Mallinger
- CDK8i compounds may be found in Mallinger et al. (Mallinger et al., 2015) (e.g. for CCT251545); Mallinger et al. (Mallinger et al., 2016) (e.g. for CCT251921 or Compound 42 / MSC2530818); Czodrowski et al., (Czodrowski et al., 2016)(e.g. for MSC2530818); Clarke et al. (Clarke et al., 2016)_(e.g. for CCT251921 or MSC2530818); all of which are herein incorporated by reference.
- CDK8 inhibitors are also described in Ettl et al. (Ettl et al., 2022), which is herein incorporated by reference.
- BI1347 (PubChem CID: 132180482) has the formula:
- RVU120 (SEL120-34A, PubChem CID: 73776233) has the formula:
- Senexin A (PubChem CID: 56927063) has the formula:
- BCD-115 (Senexin B, PubChem CID: 71661259) has the formula:
- Senexin C has the formula:
- Senexin C Active variants of Senexin C can be found in Zhang et al. (Zhang et al., 2022), which is herein incorporated by reference.
- MSC2530818 (PubChem CID: 118879529)has the formula:
- 16-didehydro-cortistatin A has the formula:
- JH-XVI-178 has the formula:
- JH-XI-10-02 (PubChem CID: 133081965) has the formula:
- the CDK8i is a nucleic acid, such as an inhibitory RNA molecule.
- the CDK8i may be an siRNA capable of inhibiting the expression of CDK8, such as inhibiting the transcription of the gene encoding CDK8 or inhibiting translation of mRNA encoding CDK8.
- the CDK8i such as a siRNA, may bind the sequence encoding CDK8 or a complementary sequence thereof.
- the siRNA may be 20-30 nucleotides long.
- the siRNA may comprise or consist of RNA or may comprise or consist of nucleotide analogues.
- the CDK8i may specifically bind (i.e. hybridise with) a sequence comprising or consisting of SEQ ID NO: 2.
- the CDK8i may be any CDK8 siRNA described herein.
- CCNCi The CCNC inhibitor (CCNCi)
- the CCNCi is a nucleic acid, such as an inhibitory RNA molecule.
- the CCNCi may be an siRNA capable of inhibiting the expression of CCNC, such as inhibiting the transcription of the gene encoding CCNCi or inhibiting translation of mRNA encoding CCNCi.
- the CCNCi such as a siRNA, may bind the sequence encoding CCNC or a complementary sequence thereof.
- the siRNA may be 20-30 nucleotides long.
- the siRNA may comprise or consist of RNA or may comprise or consist of nucleotide analogues.
- the CCNCi may specifically bind (i.e. hybridise with) a sequence comprising or consisting of SEQ ID NO: 3
- the CCNCi may be any CCNC siRNA described herein.
- inhibitors are specific examples of compounds that are known to inhibit CDC7 and/or CDK8. Derivatives and variants of these specific compounds are likely to work in a similar manner to these compounds, even if their activity is not optimal.
- Reference to a particular compound also includes variants where in one or more tertiary amine of the CDC7i and/or CDK8i (i.e. amines that are bonded to no hydrogen atoms) the N atom is replaced by P.
- a reference to a particular CDC7i and/or CDK8i compound also includes variants where in one or more secondary and/or primary amine(s) of the compound (i.e. amines that are bonded to one or two hydrogen atoms) the N atom is replaced by O, S or P.
- Reference to a particular compound also includes variants where primary amines and phosphines are functionalised by one or two R Z groups, and/or where secondary amines and phosphines are functionalised by one R Z group.
- Reference to a particular compound also includes variants where alcohols and/or ethers present in the compounds are replaced by NH, NR Z , S, PH or PR Z .
- Reference to a particular compound also includes variants where halogens (e.g. F, Cl, Br and I) are replaced with another halogen (e.g. F, Cl, Br or I), H, CN, NH 2 , NHR Z NR z 2 , PH 2 , PHR Z , PR Z 2 , OH, OR z SH or SR Z .
- Reference to a particular compound also includes variants where a nitrile group is replaced with a halogen (e.g. F, Cl, Br or I), H, OH, NH 2 , NHR Z , NR Z 2 , PH 2 , PHR Z , PR Z 2 , OR z or SR Z .
- Reference to a particular compound also includes variants where one or more primary amide is substituted by one or two are functionalised by one or two R Z groups, and/or where secondary amides are functionalised by one R Z group.
- R Z represents alkyl, alkenyl and/or aryl group(s).
- R Z groups may be C1-8 alkyl, C2-8 alkenyl or C5-8 aryl group(s), for example C1-6 alkyl, C1-6 alkenyl or C5-6 aryl group(s), preferably C1-4 alkyl or C2-4 alkenyl group(s).
- Reference to a particular compound also includes variants where one or more (e.g. two or more) of the hydrogen atoms in the hydrocarbon chain of an alkyl, alkenyl and/or alkynyl groups are replaced with substituent groups.
- substituent groups e.g. 1, 2, 3 or 4 of the hydrogen atoms in the hydrocarbon chain might be replaced with substituent groups.
- the substituent groups used may be the same or may be different.
- the alkyl, alkenyl or alkynyl group may be substituted with one or more substituent groups independently selected from halo (e.g.
- hydroxyl, amino and carboxyl groups and aryl or heteroaryl groups (especially unsaturated cyclic and heterocyclic groups with 5 to 10 atoms (e.g. 6 to 10 atoms) in their ring, such as imidazolyl, thiazolyl, thienyl, phenyl, tolyl, xylyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl or naphthyl groups).
- the alkyl, alkenyl or alkynyl group may be substituted with one or more substituent groups independently selected from halo (e.g. F, Cl and Br) hydroxyl and amino and carboxyl groups.
- the alkyl, alkenyl or alkynyl group may be substituted with one or more substituent groups independently selected from aryl or heteroaryl groups, especially unsaturated cyclic and heterocyclic groups with 5 to 10 atoms (e.g. 6 to 10 atoms) in their ring, such as imidazolyl, thiazolyl, thienyl, phenyl, tolyl, xylyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl or naphthyl groups.
- the ring itself may be substituted, e.g.
- C1-6 alkyl groups such as one or two (or more) methyl or ethyl groups, as is the case in tolyl and xylyl.
- the total number of carbon atoms in each of the substituent groups is from 5 to 12.
- Reference to a particular compound also includes variants where the length of chains, such as alkyl and polyether (e.g. polyethylene glycol) chains are shortened or lengthened, for example by ten or fewer carbon atoms or polyether units, such as 6 or fewer, or 4 or fewer, or two (e.g. one) or fewer carbon atoms or polyether units.
- a methyl group shown in a particular compound may be an ethyl group in a variant of the compound.
- Reference to a particular compound also includes variants that are ionic forms, salts, solvates, conformers, isomers, tautomers, N-oxides, esters, prodrugs, isotopes and protected forms thereof, for example, as discussed below; preferably, ionic forms, or salts or tautomers or isomers or N-oxides or solvates thereof; more preferably, ionic forms, or salts or tautomers or solvates or protected forms thereof; even more preferably the salts or tautomers or solvates thereof.
- salts for example acid addition salts or, in certain cases salts of organic and inorganic bases such as carboxylate, sulphonate and phosphate salts. All such salts are within the scope of this invention, and reference to a compound includes the salt forms of the compound.
- salt forms of the compounds of the invention are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al. (Berge et al., 1977). However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compounds of the invention, also form part of the invention.
- the salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in (Stahl et al., 2011; Wermuth and Stahl, 2002). Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
- the compounds of the invention may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed.
- Acid addition salts may be formed with a wide variety of acids, both inorganic and organic.
- acid addition salts include salts formed with an acid selected from the group consisting of acetic, hydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulphonic, toluenesulphonic, methanesulphonic (mesylate), ethanesulphonic, naphthalenesulphonic, valeric, acetic, propanoic, butanoic, malonic, glucuronic and lactobionic acids.
- Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-Lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulphonic, sebacic, stearic, succinic and tartaric acids. If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO - ), then a salt may be formed with a suitable cation.
- a functional group which may be anionic e.g., -COOH may be -COO -
- suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ .
- suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
- Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
- An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
- CDC7i and/or CDK8i compounds may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person.
- Reference to a CDC7i and/or CDK8i compound includes reference to such quaternary ammonium compounds.
- CDC7i and/or CDK8i compounds containing an amine function may also form N-oxides.
- a reference herein to a CDC7i and/or CDK8i compound) that contains an amine function also includes the N-oxide. Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide.
- N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle.
- N-Oxides can be formed as described in Advanced Organic Chemistry (Teesdale-Spittle, 1993 ) and by L. W. Deady (Deady, 1977).
- the compounds of the invention may form solvates, for example with water (i.e., hydrates) or common organic solvents.
- solvate means a physical association of the compounds of the present invention with one or more solvent molecules. This physical association involves varying degrees of intermolecular, ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.
- the term "solvate” is intended to encompass both solution-phase and isolatable solvates.
- Non-limiting examples of suitable solvates include compounds of the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.
- the compounds of the invention may exert their biological effects whilst they are in solution.
- the compounds of the present invention may have one or more polymorph (crystalline) or amorphous forms and as such are intended to be included in the scope of the invention.
- CDC7i and/or CDK8i compounds may exist in a number of different isomeric (e.g. geometrically isomeric or stereoisomeric), and tautomeric forms and references to those compounds include all such forms.
- isomeric e.g. geometrically isomeric or stereoisomeric
- tautomeric forms and references to those compounds include all such forms.
- tautomeric forms include, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/enediamines, nitroso/oxime, thioketone/enethiol, and nitro/aci-nitro.
- CDC7i and/or CDK8i compounds contain one or more chiral centres, and can exist in the form of two or more optical isomers
- references to those compounds include all optical isomeric forms thereof (e.g. enantiomers, epimers and diastereoisomers), either as individual optical isomers, or mixtures (e.g. racemic mixtures) of two or more optical isomers, unless the context requires otherwise.
- one isomer (e.g. enantiomer) in a pair of isomers may exhibit advantages over the other enantiomer, for example, in terms of biological activity.
- compositions containing a CDC7i and/or CDK8i compound having one or more chiral centres wherein at least 55% (e.g. at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compound is present as a single optical isomer (e.g. enantiomer or diastereoisomer).
- at least 55% e.g. at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95
- 99% or more (e.g. substantially all) of the total amount of the compound may be present as a single optical isomer (e.g. enantiomer or diastereoisomer).
- the compounds of the invention include compounds with one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element.
- a reference to hydrogen includes within its scope 1 H, 2 H (D), and 3 H (T).
- references to carbon and oxygen include within their scope respectively 12 C, 13 C and 14 C and 16 O and 18 O.
- Reference to CDC7i and/or CDK8i compounds may include reference to prodrugs thereof, for example esters such as carboxylic acid esters and acyloxy esters of the CDC7i and/or CDK8i compounds bearing a carboxylic acid group or a hydroxyl group.
- Particular examples of ester groups include methyl, ethyl, t-butyl and phenyl esters.
- R is an acyloxy substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably a C 1-7 alkyl group.
- prodrug means any compound that is converted into a biologically active compound in vivo.
- some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester).
- the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
- the subject may be mammalian, such as human.
- the subject is a human patient afflicted with, or at risk of, cancer.
- the administration of the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be sequential, for example, immediately or within minutes, hours or days.
- the administration of the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be concurrent.
- the administration may be concurrent where the inhibitors are co-formulated.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor are administered within one day of each other. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 48 hours of each other. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 35 hours of each other. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 30 hours of each other. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 24 hours of each other. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 16 hours of each other.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 12 hours of each other.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 8 hours of each other.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 6 hours of each other.
- the CDC7 inhibitor and CDK8 inhibitor are administered within 1 hour of each other.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 1 hour apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 4 hours apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 8 hours apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 12 hours apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 18 hours apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 24 hours apart.
- the CDC7 inhibitor and CDK8 inhibitor may be administered between about 4 hours and about 48 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 8 hours and about 48 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 12 hours and about 48 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 4 hours and about 30 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 4 hours and about 24 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 8 hours and about 30 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 8 hours and about 24 hours apart.
- the B CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 12 hours and about 30 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 12 hours and about 24 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 18 hours and about 30 hours apart.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered about 24 hours apart.
- the CDC7 inhibitor may be administered before the CDK8 (and/or CCNC) inhibitor.
- the CDK8 (and/or CCNC) inhibitor may be administered before the CDC7 inhibitor.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be provided in a composition together or provided in separate compositions.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may optionally be administered in combination with another therapeutically active agent.
- Other therapeutically active agents may comprise other immune adjuvants such as lymphokines and cytokines. Examples include interferons such as alpha, beta, and gamma interferon, interleukins such as IL-2, 11-4, IL-6, IL-13 et al., colony stimulating factors, TNFs, and the like.
- Other therapeutically active agents may comprise other antitumor agents such as chemotherapeutics and cytotoxins commonly used for treating cancer, agents that inhibit angiogenesis, and the like. These additional therapeutic agents may be administered separately or in combination. These additional therapeutic agents may be co-formulated with the CDC7 inhibitor and/or CDK8 (and/or CCNC) inhibitor.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be administered locally or systemically by any method known in the art including but not limited to intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intranasal, oral or other mucosal routes. Additional routes include intracranial (for example intracisternal, or intraventricular), intraorbital, ophthalmic, intracapsular and intraspinal administration. It is envisaged that injections will be the primary route for therapeutic administration of the compositions although delivery through a catheter or other surgical tubing is also used. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal and intramuscular administration. Liquid formulations may be utilised after reconstitution from powder formulations.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be administered in a suitable, nontoxic pharmaceutical carrier, or can be formulated in microcapsules or a sustained release implant.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be administered multiple times, if desired.
- the appropriate route, formulation, and immunization schedule can be determined by one skilled in the art.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be administered with a physiologically acceptable carrier such as physiological saline.
- the composition may also include another carrier or excipient such as buffers, such as citrate, phosphate, acetate, and bicarbonate, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins such as serum albumin, ethylenediamine tetraacetic acid, sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerol and the like.
- the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be formulated in various ways, according to the corresponding route of administration.
- the administration may be a therapeutically effective amount.
- compositions of the invention are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual.
- the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
- the compositions of the invention are particularly relevant to the treatment of existing tumours, especially cancer, and in the prevention of the recurrence of such conditions after initial treatment or surgery. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980 (Remington and Osol, 1980).
- the optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.
- the combination of the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may facilitate a significant increase in survival of treated mice in a colorectal cancer COLO205 xenograft model, or alternatively a breast cancer MDA-MB-231, MDA-MB-468 or U-2-OS model.
- the combination of the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be provided to patients/subjects that have failed to respond adequately to the standard care.
- the cancer to be treated or prevented may comprise a solid tumour malignancy.
- the cancer cell may comprise or consist of a cancer cell of a solid tumour.
- the solid tumour may comprise a sarcoma, carcinoma, or lymphoma.
- the cancer may comprise neuroblastoma or melanoma.
- Solid tumors that can be treated using the compositions and methods described herein may be selected from any one of the group of solid tumors of the breast, lung, colon, stomach, liver, kidney, ovary, and prostate; or combinations thereof.
- Tumors that can be treated in accordance with the invention may be selected from breast carcinomas, lung carcinomas, prostate carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, vulval carcinomas, kidney carcinomas, cervical carcinomas, endometrial carcinoma, endometrial hyperplasia, endometriosis, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas, sarcomas include fibrosar
- the cancer to be treated or prevented may be a blood cancer, such as leukemia, lymphoma or myeloma.
- the cancer to be treated or prevented is osteosarcoma or epithelial adenocarcinoma.
- a CDC7 inhibitor in combination with a CDK8 and/or CCNC inhibitor for use in the treatment or prevention of cancer in a subject.
- composition comprising a CDC7 inhibitor and a CDK8 and/or CCNC inhibitor.
- kits for treatment or prevention of cancer in a subject comprising:
- composition or kit according to the invention for use as a medicament.
- composition according to the invention, or kit according to the invention for use in the treatment or prevention of cancer in a subject.
- a method of treatment or prevention of cancer in a subject wherein the cancer is determined or known to be deficient in functional CDK8 and/or CCNC expression, the method comprising the administration of a Cell Division Cycle 7 kinase inhibitor (CDC7i) to the subject.
- CDC7i Cell Division Cycle 7 kinase inhibitor
- a Cell Division Cycle 7 kinase inhibitor for use in a method of treatment or prevention of cancer in a subject, wherein the cancer is determined or known to be deficient in functional CDK8 and/or CNCC expression.
- the invention recognises that some subjects may have a cancer that is CDK8 deficient, which would then obviate the need for a CDK8i and/or CCNCi to be administered in combination with a CDC7i.
- cancers deficient in CDK8 and/or CNCC represent a sub-class of cancers that may be targeted for treatment more efficiently by single CDC7i therapy.
- the method may comprise the treatment of DLBCL having CDK8 deficiency with a CDC7 inhibitor (CDC7i).
- CDC7i a CDC7 inhibitor
- the method may comprise the treatment of such cancers having CDK8 deficiency with a CDC7 inhibitor (CDC7i).
- the method may comprise the treatment of such cancers having CCNC deficiency with a CDC7 inhibitor (CDC7i).
- CDK8 deficiency is understood to mean that the cells, such as the cancer cells, do not express functional CDK8, or the CDK8 expression is significantly lower than average expression levels in normal cells (i.e. non-CDK8-deficient cells), such as at least 3-fold, 5-fold, 10-fold, 50-fold or 100-fold lower.
- CCNC deficiency is understood to mean that the cells, such as the cancer cells, do not express functional CCNC, or the CCNC expression is significantly lower than average expression levels in normal cells (i.e. non- CCNC -deficient cells), such as at least 3-fold, 5-fold, 10-fold, 50-fold or 100-fold lower.
- the invention has advantageously found that tumors defective in either CDK8 and/or CCNC can be particularly sensitive to CDC7i-basesd chemotherapy.
- CDK8 and/or CCNC as biomarkers for predicting the efficacy of CDC7i therapy for cancer of a subject, wherein the reduction or absence of expression of functional CDK8 and/or CCNC in a subject is indicative of an increased efficacy of the CDC7i therapy for cancer.
- a method of predicting the efficacy of a CDC7i therapy for cancer in a subject comprising determining the levels of expression of functional CDK8 and/or CCNC in the subject, or obtaining results of such a determination, wherein a decreased level of expression of CDK8 and/or CCNC in the subject is indicative of a more effective CDC7i therapy for the cancer in the subject.
- a method of selecting a subject for treatment for cancer with a CDC7i comprising determining the levels of expression of functional CDK8 and/or CCNC in the subject, or obtaining results of such a determination, wherein the subject is selected for CDC7i treatment if they have a decreased level of expression of CDK8 and/or CCNC.
- the methods or uses of the invention may further comprise the treatment of the subject with a CDC7i, for example by administration of a CDC7i to the subject.
- a method of treatment of a subject for cancer comprising:
- the increased efficacy may be relative to cancer subjects having normal or higher levels of expression of functional CDK8 and/or CCNC.
- the increased efficacy may be a statistically significant increase in efficacy.
- the decreased level of expression of CDK8 and/or CCNC may be relative to a reference level.
- the reference level may be an average level of expression in a general population, or an average level in a population of subjects having cancer, preferably the same cancer type and/or stage.
- the reduction or absence of expression of functional CDK8 and/or CCNC in a subject may be in a selected tissue, such as tumour tissue or cells.
- a selected tissue such as tumour tissue or cells.
- the tumour tissue or cells of the subject may be determined for their expression of functional CDK8 and/or CCNC.
- the reduction/decreased level of expression of functional CDK8 and/or CCNC may be a 100% decrease.
- the decreased level of expression of CDK8 and/or CCNC may be a 50%, 80%, 90%, 95% or 99% decrease.
- the CDC7i may be administered and/or formulated as described herein.
- the CDC7i may be a The CDC7i described herein.
- inhibitor is understood to refer to an agent that is capable of preventing or reducing the activity of a target molecule, or a biological pathway.
- the inhibition may be complete, i.e. 100% inhibition, or the inhibition may be partial.
- the inhibition may inhibit up to 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the original activity of the target (i.e. relative to the activity when not in the presence of the inhibitor).
- the inhibition may be sufficient to effect a statistically significant reduction in activity of the target.
- the level of inhibition is sufficient to cause a therapeutic effect.
- subject means all animals including humans. Examples of subjects include humans, cows, dogs, cats, goats, sheep, and pigs.
- patient means a subject having a disorder in need of treatment.
- a 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity.
- Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent.
- a therapeutically effective amount of the active component is provided.
- a therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
- the term "functional” is understood to refer to the normal function of a given biological molecule such as CDC7, CDK8 or CCNC.
- a loss of function may be complete, such that it cannot carry out its normal ligand binding and/or reaction, or it may have a significant impairment in activity, such as a lower ability to bind its ligand or a lower rate of reaction.
- a loss of "function” or “non-functional molecule” may be a loss of the activity of the molecule, or a lack of expression of the molecule.
- a panel of different cell lines including: MDA-MB-468 (a - c); MDA-MB-231 (d - f); and U-2 OS (g - i), were mock treated or treated with 1.25 ⁇ M XL413 and/or 31 nM BI-1347BI-1347 for 24 hours.
- Cells were labelled with EdU for last 30 minutes and DNA synthesis, DNA content and cell cycle distribution were assessed by flow cytometry and analysed using the gating strategy described in Figure 2c .
- Eukaryotic DNA replication initiates at multiple origins according to a well-defined spatiotemporal program, with origins being activated in early, mid-, or late S phase, each one giving rise to two replication forks (Gilbert et al., 2010). Origin activation is orchestrated by the coordinate action of Cyclin-dependent kinases (CDKs) and CDC7 kinase, also known as DDK (DBF4-dependent kinase).
- CDKs Cyclin-dependent kinases
- DDK DVF4-dependent kinase
- CDC7is small-molecule ATP-competitive CDC7 inhibitors
- CDC7 is an essential gene in almost every cell line tested thus far (Cowley et al., 2014; McFarland et al., 2018; Meyers et al., 2017; Rainey et al., 2017; Tsherniak et al., 2017), it is now clear that different cancer cells have a wide range of sensitivity to either partial or transient CDC7 inhibition (Iwai et al., 2019; McDonald et al., 2017; Sasi et al., 2014). This suggests that multiple factors may determine how cells respond to decreased levels of CDC7 activity and that the response to limited origin firing is genetically controlled.
- CDK8 is a ubiquitously expressed nuclear Ser/Thr kinases, that is a component of the "Mediator-CDK8 module", and is traditionally classified as a “transcriptional” rather than a “cell cycle” CDK (Dannappel et al., 2019; Galbraith et al., 2010).
- the Mediator is a large multi-subunit complex, consisting of a "head”, “middle”, and “tail” at the core of its structure, which is essential for its function in transcription by bridging transcriptional elements, promoters, chromatin modifiers and transcription factors (TFs) to RNA polymerase II (Fant and Taatjes, 2019; Harper and Taatjes, 2018).
- CDK8 module is a 4 subunit regulatory subcomplex in which Cyclin C (CCNC) and MED12, activate CDK8 kinase function, and MED13, enables their interaction with the Mediator (Dannappel et al., 2019; Fant and Taatjes, 2019; Menzl et al., 2019).
- CDK19 in mammals a paralog of CDK8, namely CDK19, exists and has high sequence similarity to CDK8.
- CDK19 in a mutually exclusive manner with CDK8 can form an analogous "CDK19 module" (Tsutsui et al., 2008).
- MED12L MED12-like
- MED13L MED13-like
- CDK8 primarily regulates cellular function through phosphorylation of a large number of proteins including: Mediator components, TFs; chromatin remodelers, and RNA polymerase II, in addition, to non-mediator associated proteins (Poss et al., 2016).
- CDK8 activity is not required for bulk transcription, but it regulates the expression of subsets of genes in cell type- and context-specific manner. This uniquely highlights CDK8 as mediators of transcriptional reprogramming, rather than regulators of global transcription, like CDK7 and CDK9 (Chen et al., 2019; Fant and Taatjes, 2019; Nemet et al., 2014).
- CDK8 was first described as a proto-oncogene in colorectal cancer (CRC), where high expression levels negatively correlated with poor patient survival and were associated with advance stage disease progression (Firestein et al., 2008, 2010; Seo et al., 2010).
- CRC colorectal cancer
- downregulation of CDK8 reduced proliferation of CRC cells in vitro and in xenograft models with CDK8 amplification, highlighting the therapeutic potential of targeting CDK8 (Adler et al., 2012; Firestein et al., 2008; Seo et al., 2010).
- CDK8 inhibitors have now been developed as targeted agents and because of the similarity between CDK8 and CDK19 active site most of these compounds can be considered as dual CDK8/19 inhibitors (Fant and Taatjes, 2019; Philip et al., 2018; Xi et al., 2019). Some of these have shown cancer type-specific therapeutic effects in in vivo colorectal (Liang et al., 2018), breast (McDermott et al., 2017), leukemia (Pelish et al., 2015; Rzymski et al., 2017), prostate (Nakamura et al., 2018) and lung (Porter et al., 2012) models.
- CDK8 signalling was also shown to modulate the cytotoxic effects of NK cells, promoting tumour surveillance and increasing survival in mouse melanoma, leukemia and metastasising breast cancer models (Hofmann et al., 2020; Putz et al., 2013).
- CDK8 inhibitors could be mediated by both cell-intrinsic mechanisms, leading to reduced tumour cell proliferation and survival, as well as cell-extrinsic mechanism mediated by the host immune system and tumor microenvironment.
- CDK8 inhibition is a potentially promising therapeutic approach, however, as in the case of CDC7is, the development of CDK8 based therapy has obstacles to overcome. For example tolerability issues have been observed with some inhibitors (Chen et al., 2019) and as a result only select inhibitors, RVU120 and BCD-115, have progressed to clinical trials targeting acute myeloid leukemia (ClinicalTrials.gov Identifier: NCT04021368) and estrogen-receptor positive breast cancers (ClinicalTrials.gov Identifier: NCT03065010), respectively.
- CDK8i based therapy Due to the cell-type specific regulation of gene expression by CDK8, the best indication for a CDK8i based therapy has not yet been identified and maybe limited to only a few cancer types/subtypes. Finally, all CDK8 inhibitors identified so far also target CDK19, and developing monospecific CDK8 inhibitors could be a challenge due to the high sequence similarity shared between CDK8 and CDK19 (Philip et al., 2018; Tsutsui et al., 2008).
- CDK8/CDK19-CCNC can bind to the human homolog of yeast sld7 protein, MTBP, an essential DNA replication origin firing factor (Boos et al., 2013). This association occurs independently from MED12 and MED13, suggesting this could represent a role independent from the Mediator complex and transcription (Köhler et al., 2019). This interaction is mediated by a metazoan specific binding region in MTBP that is important for efficient DNA synthesis, preventing replication stress and ensuring complete genome duplication to support a normal mitosis (Köhler et al., 2019).
- CDK8/CDK19-CCNC can bind and phosphorylate MTBP in vitro
- CDK8 dependent phosphorylation of MTBP in living cells has yet not been detected nor studied (Ferreira et al., 2021; Köhler et al., 2019).
- a genome-wide CRIPSR/Cas9 loss of function screen identifies genes that affect proliferation rate in response to a CDC7 inhibitor
- MCF10A EditR cells that stably express Cas9 were transduced with a lentiviral-sgRNA library targeting approximately 19,000 coding genes and then were either untreated or treated with 1.25 ⁇ M of selective ATP competitive CDC7 inhibitor, XL413 (Koltun et al., 2012).
- CRISPR/Cas9-mediated loss of function of genes involved in supporting proliferation in untreated cells or in response to CDC7 inhibition is expected to result in a decreased relative abundance of the corresponding sgRNAs in the cell population.
- CDK8 a gene that expresses a component of the CDK8/CDK19-CCNC-MED 12-MED 13 mediator complex kinase-module (Dannappel et al., 2019; Fant and Taatjes, 2019; Menzl et al., 2019), came out as the top hit ( Figure 1b and Table 1, ranked 19012 out of 19012 genes).
- Table 1 The table summarises the beta scores, assigned by MAGeCK-MLE, and based on changes of sgRNA abundance from day 0 to day 20 or 24 in untreated or XL413-treated samples, respectively.
- Genes of interest are shown in rank order, by the differential beta score, alongside the number of sgRNAs used to target each gene, Uniprot identifier (Uniprot ID), protein name and functional annotation.
- Significant gene outliers were identified using thresholds of +/- 2 standard deviations from the mean differential of the beta score (Treatment - Control).
- ( ⁇ 1 ) and ( ⁇ 2 ) represent candidate gene hits, which upon editing, either significantly enhance or suppress the anti-proliferative effects of XL413, respectively.
- Rank Gene XL413 beta score Untreated beta score Differential beta score Uniprot ID Uniprot protein name and functional annotation 1 ( ⁇ 1 ) RIF1 - 0.0162 -0.7603 0.7442 Q5UIP0 Replication timing regulatory factor 1; Telomere-associated protein RIF1; Key regulator of TP53BP1 that plays a key role in the repair of double-strand DNA breaks (DSBs) in response to DNA damage: acts by promoting non-homologous end joining (NHEJ)-mediated repair of DSBs.
- NHEJ non-homologous end joining
- RNA polymerase II transcription subunit 12 Component of the Mediator complex, a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes.
- Mediator functions as a bridge to convey information from gene-specific regulatory proteins to the basal RNA polymerase II transcription machinery.
- Mediator is recruited to promoters by direct interactions with regulatory proteins and serves as a scaffold for the assembly of a functional preinitiation complex with RNA polymerase II and the general transcription factors.
- RNA polymerase II transcription subunit 13 Component of the Mediator complex, a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes.
- NB Mediator function and recruitment as described for MED12 . 10184 CDK19 - 0.2393 -0.2427 0.0034 Q9BWU1 Cyclin-dependent kinase 8/11; Cyclin dependent kinase 19; Belongs to the protein kinase superfamily.
- NB Mediator function and recruitment as described for MED12 . 19012( ⁇ 2 ) CDK8 - 1.6094 -0.1703 -1.4391 P49336 Cyclin-dependent kinase 8; Component of the Mediator complex, a coactivator involved in regulated gene transcription of nearly all RNA polymerase II-dependent genes. Phosphorylates the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNAp II), which may inhibit the formation of a transcription initiation complex.
- NB Mediator function and recruitment as described for MED12.
- CCNC the cognate cyclin of CDK8
- CDK8 paralog, CDK19, and other components of the mediator complex kinase-module, MED12 and MED13 were not required.
- ⁇ 96 genes which upon loss of function, could increase the rate of proliferation specifically in cells treated with low dose CDC7i.
- RIF1 a gene we have showed could reduce the requirement for CDC7 kinase when deleted in our previous CRISPR/Cas9 screen study (Rainey et al., 2020), was once again ranked as the top hit in this gene cohort and instils confidence in the results of this screen. ( Figure 1b and Table 1, ranked 1 out of 19012 genes).
- CDK8 showed significant and robust decreases in accumulation of multiple sgRNAs, but only under conditions of limited CDC7 activity (Supplemental Figure 2a ).
- CDK19 which shows 76% sequence identity (Supplemental Figure 2f ), and which is also regulated by CCNC, does not strongly or reproducibly affect sgRNA abundance between the untreated and CDC7i treated branches (Supplemental Figure 2b ).
- CDK8/CDK19 determine the potency of CDC7 inhibitors in proliferation and DNA replication assays
- CDK8/CDK19-CCNC complex was shown to bind and phosphorylate the human replication initiation factor, MTBP, in vitro (Ferreira et al., 2021; Köhler et al., 2019). While the region of MTBP, required for binding CDK8/CDK19-CCNC complex, is important for normal genome replication, the requirement and role of CDK8/CDK19-CCNC in this process is poorly understood (Ferreira et al., 2021; Köhler et al., 2019). For this reason, we focused our analysis on determining their role in the response to CDC7 inhibition.
- CDK8/CDK19 inhibition resulted in a dose-dependent increase in G1 phase cells with a corresponding decrease in early-mid S phase cells.
- a more refined analysis however we noticed a subtle and dose-independent decrease in the rate of DNA synthesis in S phase cells ( Figure 2f ).
- Strikingly, in XL413 treated cells inhibition of CDK8/CDK19 resulted in a further accumulation of mid-late S phase cells with a profound and dose independent decrease in the rate of DNA synthesis ( Figure 2c-f ). Together these results indicate CDK8/CDK19 activity is required to promote efficient DNA synthesis and such role in S-phase becomes critical when CDC7 activity becomes rate limiting.
- CDK8 deletion is present in sarcomas ( ⁇ 4.31 % of 255 cases), lung adenocarcinomas ( ⁇ 2.47 % of 566 cases), and diffuse large B-cell lymphoma ( ⁇ 2.08 % of 48 cases).
- CCNC deletions instead is observed at high rate in diffuse large B-cell lymphoma ( ⁇ 12.5 % of 48 cases) prostate adenocarcinoma ( ⁇ 9.11 % of 494 cases) uveal melanoma ( ⁇ 7.5 % of 80 cases), liver hepatocellular carcinoma ( ⁇ 3.23 % of 372 cases) and stomach adenocarcinoma ( ⁇ 2.95 % of 440 cases), that show frequent deep of CCNC.
- CDK8 and CCNC are potential novel biomarkers whose functional status can modulate the anti-cancer effects of CDC7i monotherapy. Furthermore we show, for the first time, that inhibition of CDK8/CDK19 potentiates the anti-proliferative activity a of CDC7 inhibition suggesting that a combination of CDC7 with CDK8 inhibitors may improve efficacy and can be potentially efficacious across a broader spectrum of cancers when compared to either monotherapy.
- MCF10A cell line human, female origin
- MCF10A-EditR cells stably expressing Cas9 were generated as previously described (Rainey et al., 2017).
- MCF10A cells and derivatives were cultured using DMEM supplemented with 5% horse serum, 25 ng/ml cholera toxin, 10 ⁇ g/ml insulin, 20 ng/ml epidermal growth factor (Peprotech), 500 ng/ml hydrocortisone, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin.
- Lenti-X TM 293T cell line human, female origin
- Routine culture was performed using DMEM +GlutMAX TM -I (ThermoFisher Scientific) supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin.
- MDA-MB-468 human, female origin
- RPMI-1640 medium Sigma-Aldrich
- MDA-MB-231 human, female origin
- DMEM fetal bovine serum
- a plasmid based CRISPR pooled lentiviral sgRNA library that can be used in a modular format was purchased from CELLECTA (CELLECTA: CRISPR Human Genome Knockout Library, Module 1 (200 ⁇ g plasmid), #sgRNA: 52,549, KOHGW-M1-P; Module 2 (200 ⁇ g plasmid), #sgRNA: 49,461, KOHGW-M2-P; and Module 3 (200 ⁇ g plasmid), #sgRNA: 48,578, KOHGW-M3-P). Each module covers approximately 6,300 genes and each gene is targeted by 8x sgRNAs ( ⁇ 50,000 sgRNAs per module), in total targeting >19,000 protein coding genes.
- Lenti-X 293T cells 25 ⁇ 10 6 Lenti-X 293T cells were seeded on 20 ⁇ 150 mm plates for each sgRNA library module and transfected the following day.
- a master stock of packaging plasmid (600 ⁇ g) and sgRNA plasmid (120 ⁇ g) were combined in 20 ml of 150 mM NaCl and mixed with 1440 ⁇ l of polyethylenimine (1 mg/ml) that had been diluted in 20 ml of 150 mM NaCl. After 20 minutes of incubation 2 ml of the mix were added dropwise to each plate.
- MCF10A EditR cells Aliquots of viral supernatant were titered by adding incremental volumes (0.1 ⁇ l to 1 ⁇ l for concentrated virus or 10 ⁇ l to 100 ⁇ l for viral supernatant) in the presence of polybrene (5 ⁇ g/ml) to MCF10A EditR cells that had been seeded 24 hours in advance at 120,000 cells per well in a 6 well plate. Media was changed after 24 hours and at 48 hours after transduction, MCF10A EditR cells were harvested and fixed in 4% paraformaldehyde in PBS for 20 minutes at room temperature before being washed and resuspended in PBS.
- MCF10A EditR cells 120 ⁇ 10 6 per module were transduced with individual modules (Modules I, II and III) in the presence of 5 ⁇ g/ml polybrene at a Multiplicity of Infection (Mol) of 0.4 achieving ⁇ 600-fold guide representation. After 24 hours, transduced cells were harvested, replated and selected with puromycin (1 ⁇ g/ml) for 6 days. Puromycin resistant cells were harvested and a sample of ⁇ 30 ⁇ 10 6 cells taken for analysis by next generation sequencing (NGS) at the start of the screen (day 0). The remaining puromycin resistant cells from each module were cultured independently from each other in untreated and XL413 treated (1.25 ⁇ M) experimental branches.
- NGS next generation sequencing
- Cell pellets were resuspended in 6 ml of Buffer P1 (50 mM Tris-Cl, pH 8.0, 10 mM EDTA, Qiagen) and lysed by addition of SDS to final concentration of 1 % (v/v). Samples were sonicated at 10 % amplitude for 5 seconds (Branson, digital sonifier) before addition of proteinase K (40 ⁇ g/ml, Qiagen) followed by gentle mixing and incubation at 55°C overnight. Samples were cooled to 37°C, and following addition of RNaseA (100 ⁇ g/ml, Qiagen), incubated for 30min.
- Buffer P1 50 mM Tris-Cl, pH 8.0, 10 mM EDTA, Qiagen
- SDS final concentration of 1 % (v/v). Samples were sonicated at 10 % amplitude for 5 seconds (Branson, digital sonifier) before addition of proteinase K (40 ⁇
- Genomic DNA was subject to a first round PCR using multiple reactions, with a maximum of 50 ⁇ g of genomic DNA in each 100 ⁇ l PCR reaction, with 0.2 mM of each dNTP, 0.3 ⁇ M of each primer (Forward 5'-AGGGCCTATTTCCCATGATTCCT-3' and Reverse 5'-CGACAACAACGCAGAAATTTTGAAT-3'), and 1x Titanium Taq DNA polymerase in 1x Titanium Taq buffer. Genomic DNA was initially denatured at 94°C for 4 minutes, followed by 18 cycles of 94°C for 45 seconds, 63°C for 15 seconds, 72°C for 35 seconds, 1 cycle of 72°C for 4 minutes and hold at 4°C.
- the 2 nd round PCR was subject to PCR clean-up (Macherey-Nagel), to concentrate the DNA into a 50 ⁇ l volume, followed by gel purification (Macherey-Nagel) of a DNA product of ⁇ 293bp from a 2 % (w/v) agarose DNA gel.
- Gel purified libraries were sent to Novagene for ilummina NGS analysis using a NovaSeq 6000 PE150, paired-end reads and 30Gb of raw data per sample.
- the libraries were shipped to Novagene for ilummina NGS analysis using a NovaSeq 6000 PE150, with paired-end reads and 30 Gb raw data per sample.
- MAGeCKFlute pipeline Wang et al., 2019
- MAGeCK was used to perform adaptor trimming, mapping of trimmed sgRNAs and associated sequencing reads to the CELLECTA sgRNA reference library, and a quality control assessment of the sequencing quality and screen data.
- sgRNA read count data from the day 0 vs day 20 untreated and day 0 vs day 24 XL413-treated (1.25 ⁇ M) conditions were modelled using a negative-binomial distribution by MAGeCK-MLE to calculate a beta score for each targeted gene.
- Negative control sgRNAs in the CELLECTA library were used by MAGeCK-MLE to normalize the read counts between samples.
- Beta score normalization was performed by MAGeCK-Flute, using the proposed list of core essential genes (Wang et al., 2019), to accommodate the effect of the CDC7 inhibitor on decreased cell proliferation throughout the screen. No copy number correction was applied as the MCF10A EditR cell line used in the screen are near diploid.
- the normalized control beta score was subtracted from normalized treatment beta score, for each gene target, and the genes were ranked and plotted based on this differential beta score. Gene hits were identified as outliers +/- 2x standard deviation of the mean differential beta score. For the hits identified, the normalised sgRNA count data was plotted and used to compare the relative abundance in day 20 untreated and day 24 XL413-treated (1.25 ⁇ M) samples to day 0.
- MCF10A cells were seeded at 4,000 cells per well of a 6 well plate and after 24 hours were treated with inhibitors before being incubated for 7 days at 37 °C with 5% CO 2 . Media was removed from the cells which were then washed with PBS and air dried before being fixed with 100 % methanol for 1 hour. Following removal of methanol the plates were air-dried, stained with crystal violet staining solution (0.2% crystal violet, 25% methanol) for 1 hour at room temperature, washed with ddH 2 O and air-dried. Plates were scanned using a HP Scanjet 4050, to generate images of crystal violet stained cells, and crystal violet staining intensity was acquired using the Odyssey infrared imaging system and Image Studio software (LI-COR Biosciences).
- nascent DNA was labelled by incubating cells with 10 ⁇ M EdU for either 20 or 30 minutes prior to harvest, washing with PBS, and then by resuspension in PBS containing 70% EtOH for 1 hour at -20 °C. After a PBS wash, incorporated EdU was labelled by incubating cells in click reaction buffer (PBS containing 10 ⁇ M 6-Carboxyfluorescine-TEG-azide, 10 mM Sodium-L-ascorbate, 2 mM Copper-II-Sulphate) for 30 minutes protected from light.
- click reaction buffer PBS containing 10 ⁇ M 6-Carboxyfluorescine-TEG-azide, 10 mM Sodium-L-ascorbate, 2 mM Copper-II-Sulphate
- Fluorescence intensity data for 6-Carboxyfluorescine (488_530_30 nm) and DAPI (405_450_50 nm) were acquired for 10,000 single cells on a BD FACS Canto II and analysed using FlowJo software.
- fluorescence intensity, proportional to EdU incorporation, for individual cells gates were applied on DAPI-EdU biparametric dot plots to select for EdU positive S phase cells using FlowJo. According to the required analysis these gates included either all S phase cells, or only early or late S phase cells.
- Fluorescence intensity (488_530_30) values per cell were exported and plotted as scatter dot plots using GraphPad prism.
- mRNA sequences below represent the longest transcripts and encode the longest isoforms for: CDC7 (NM_003503.4), CDK8 (NM_001260.3) and CCNC (NM_005190.4), respectively. In each case the coding sequences is highlighted by an underline.
- CDC7 Homo sapiens cell division cycle 7 (CDC7), transcript variant 1, mRNA (SEQ ID NO: 1)
- CDK8 Homo sapiens cyclin dependent kinase 8 (CDK8), transcript variant 1, mRNA (SEQ ID NO: 2)
- NCBI reference sequence NM_001260.3:
- CCNC Homo sapiens cyclin C
- SEQ ID NO: 3 Homo sapiens cyclin C
- SEQ ID NO: 3 Homo sapiens cyclin C (CCNC), transcript variant 1, mRNA (SEQ ID NO: 3)
- Example target sequences for siRNA oligos specific for Cdc7 coding region include, but is not an exhaustive list: 5'-aagctcagcaggaaaggtgtt-3' (Cdc7-A); 5'-aaagaagttccagcacaagac-3' (Cdc7-B); and 5'-aagcagtcaaagactgtggat-3' (Cdc7-D) (Montagnoli et al., 2004).
- CDK8 and CCNC siRNA CDK8 and CCNC siRNA
- Example target sequences for siRNA oligos specific for CDK8 and CCNC coding region include, but is not an exhaustive list: 5'-GCAACCAUUCUUAGUAAGAUGCCAA-3' (CCNC #1), 5'-GGAAUUUCCUUAUAGGAUGAAUCAT-3' (CCNC #2), 5'-AGGCAUUAUACCAAACGUAUUGATA-3' (CDK8 #1), 5'-GUAAAGCAUUCCACUUGCUUCAGAA-3' (CDK8 #2) (Lloyd et al., 2021).
- Example target sequences for lentiviral shRNA constructs generated by the RNAi consortium (GPP Web Portal - Home (broadinstitute.org)). Some of these shRNA sequences have been used to target CDK8 and CCNC (Firestein et al., 2008) and are also commercially available through sigma-aldrich/Merck. This is not an exhaustive list.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a combination therapy. In particular, a Cell Division Cycle 7 kinase inhibitor (CDC7i) used in a combination therapy with: i) a Cyclin Dependent Kinase 8 inhibitor (CDK8i), and/or ii) a Cyclin C inhibitor (CCNCi); for use in the treatment or prevention of cancer in a subject.
Description
- The present invention relates to a combination therapy to treat or prevent cancer, including methods of treatment or prevention, uses, compositions and kits for treating cancer in a subject.
- During tumorigenesis, oncogene activation results in dysregulation of DNA replication and transcription programmes ultimately driving the hyper-proliferative state of cancer cells. Cell Division Cycle 7 (CDC7) kinase encoded by the CDC7 gene is considered to be a key switch for DNA replication and its depletion by siRNA causes cancer cells to stop DNA synthesis. As such, a number of CDC7 kinase inhibitors (termed "CDC7i" herein) are in development as a novel class of anti-cancer treatment. Such clinical studies include (ClinicalTrials.gov):
- 1. Study of BMS-863233 in patients with hematologic cancer: Terminated
- 2. Study of BMS-863233 in patients with advanced and/or metastatic solid tumors: Terminated
- 3. CR-UK phase I trial of LY3143921: Recruiting
- 4. Clinical study of TQB3824 in subjects with advanced cancer: Recruiting
- 5. Study of NMS-1116354 in solid tumors: Terminated
- 6. Study of NMS-1116354 in advanced/metastatic solid tumors: Terminated
- 7. Study to evaluate TAK-931 in participants with advanced nonhematologic tumors: completed
- However, problems persist in this approach where there are ongoing issues with reaching a good level of efficacy whilst avoiding toxicity of these CDC7 kinase inhibitors. In particular, tolerability issues have been observed with some inhibitors leading to their clinical trial termination.
- Most likely CDC7 inhibitors will be included as a component of chemotherapy with other agents. Previous studies have shown that inhibition of the ATR kinase can modify the cell sensitivity to CDC7i, however enhancement of CDC7i anti-cancer activity is observed only at very high doses of CDC7i while suppression is observed at lower doses (Guo et al., 2021; Iwai et al., 2019; Rainey et al., 2020). Such a combination therapy does not adequately address the clinical need.
- Thus, there is a clear clinical need for more novel therapeutic agents for cancer treatment and an ongoing need to find alternative and improved therapies relative to those under study.
- An aim of the present invention is to provide an alternative or improved therapy for cancer.
- According to a first aspect of the invention there is provided a method of treatment or prevention of cancer in a subject comprising the administration of a Cell Division Cycle 7 kinase inhibitor (CDC7i) in combination with:
- i) a Cyclin Dependent Kinase 8 inhibitor (CDK8i), and/or
- ii) a Cyclin C inhibitor (CCNCi).
- The invention advantageously identifies CDK8 as a protein, that assists CDC7 in DNA replication. It is found that pharmacological inhibition of CDK8 has a negligible effect on DNA replication and cell division, but greatly enforces the anti-proliferative activity of CDC7 inhibitors when used in combination. Thus, the use of CDK8 inhibitors (and/or inhibitors of its cognate CCNC) in combination with CDC7 inhibitors greatly improve the efficacy of the latter anti-cancer drug. CDK8 inhibitors are being developed as potential anti-cancer agents with 2 reported clinical trials (ClinicalTrials.gov):
- 1. Study of BCD-115 in women with ER(+) HER2(-) local advanced and metastatic breast cancer: completed (treatment discontinuation due to adverse events)
- 2. RVU120(SEL120) in patients with acute myeloid leukaemia or high-risk myelodysplastic syndrome: Phase I; recruiting.
- However, despite the typical monotherapeutic approach for treatment of cancer, the present invention finds for the first time that CDC7i and CDK8i (additionally or alternative CCNCi) synergise in stopping DNA replication and cell proliferation.
- Therefore, combination therapy using CDC7i and CDK8i can be more efficacious and less toxic than CDC7 inhibitor monotherapy for the treatment of solid cancers, blood cancers and other proliferative diseases.
- The CDC7i may bind to CDC7. In one embodiment, the binding to CDC7 is preferential or specific binding.
- The CDC7i may be a small molecule (i.e. less than 900Da). The CDC7i may be a small molecule ATP-competitive CDC7 inhibitor,
- The CDC7i may comprise or consist of XL413, or a functional derivative or variant thereof having CDC7 inhibition activity.
- The CDC7i may comprise or consist of any one of the group of CDC7 inhibitors selected from PHA-767491 (Montagnoli et al., 2008), XL413 (BMS-863233; Clinicaltrials.gov ID: NCT00838890 or NCT00886782) (Koltun et al., 2012), AS-0141 (Irie et al., 2021), Dequalinium Chloride, Clofoctol (Cheng et al., 2018), LY3143921 (Clinicaltrials.gov ID: NCT03096054), TQB3824 (Clinicaltrials.gov ID: NCT05028218), NMS-1116354 (Clinicaltrials.gov ID: NCT01016327 or NCT01092052) and TAK-931 (Clinicaltrials.gov ID: NCT02699749) (Iwai et al., 2019); or combinations thereof.
-
- Active variants of PHA-767491 can be found in Vanotti et al. (Vanotti et al., 2008), Menichincheri et al. (Menichincheri et al., 2009), Ermoli et al. (Ermoli et al., 2009), and Menichincheri et al. (Menichincheri et al., 2010), which are herein incorporated by reference.
-
- Active variants of XL413 can be found in Koltun et al. (Koltun et al., 2012), which is herein incorporated by reference.
-
- Active variants of AS-0141 can be found in Irie et al. (Irie et al., 2017); and Irie et al. (Irie et al., 2021), which are herein incorporated by reference.
-
-
-
-
-
-
- TAK-931 is described in Iwai K et al. (Iwai et al., 2019), which is herein incorporated by reference. Active variants of TAK-931 may be found in Kurasawa et al. (Kurasawa et al., 2020) and Kurasawa et al. (Kurasawa et al., 2017), both of which are herein incorporated by reference.
- In a preferred embodiment, the CDC7i comprises or consists of TAK931 or AS-0114. In one embodiment, the CDC7i comprises or consists of TAK931. In another embodiment, the CDC7i comprises or consists of AS-0114. In another preferred embodiment, the CDC7i comprises or consists of XL413 (BMS-863233).
- The skilled person will recognise that functional derivatives or variants of these CDC7i molecules may be available for use in the invention.
- In another embodiment, the CDC7i is a nucleic acid, such as an inhibitory RNA molecule. The CDC7i may be an siRNA capable of inhibiting the expression of CDC7, such as inhibiting the transcription of the gene encoding CDC7 or inhibiting translation of mRNA encoding CDC7. The CDC7i, such as a siRNA, may bind the sequence encoding CDC7 or a complementary sequence thereof. The siRNA may be 20-30 nucleotides long. The siRNA may comprise or consist of RNA or may comprise or consist of nucleotide analogues. The CDC7i may specifically bind (i.e. hybridise with) a sequence comprising or consisting of SEQ ID NO: 1. The CDC7i may be any CDC7 siRNA described herein.
- The CDK8i may be a dual CDK8/CDK19 inhibitor. In another embodiment, the CDK8i inhibitor may be specific for CDK8 and not CDK19. The CDK8i may not significantly inhibit CDK19.
- The CDK8i may comprise or consist of BI1347, or a functional derivative or variant thereof having CDK8 inhibition activity.
- The CDK8i may comprise or consist of any one of the group of CDK8 inhibitors selected from BI1347 (Hofmann et al., 2020), RVU120 (SEL120-34A, Clinicaltrials.gov ID: NCT04021368) (Johannessen et al., 2017), Senexin A (Porter et al., 2012), BCD-115 (Senexin B, Clinicaltrials.gov ID: NCT03065010) (Johannessen et al., 2017), Senexin C (Zhang et al., 2022), MSC2530818 (Czodrowski et al., 2016; Mallinger et al., 2016), AS2863619 (Akamatsu et al., 2019), 16-didehydro-cortistatin A (DCA) (Arnett et al., 2021), BRD6989 (Johannessen et al., 2017), CCT-251545 (Mallinger et al., 2015), CCT-251921 (Mallinger et al., 2016), TSN084 (Clinicaltrials.gov ID: NCT05300438), JH-XVI-178 (Hatcher et al., 2021), and JH-XI-10-02 (Hatcher et al., 2018); or combinations thereof.
- Active variants of the CDK8i compounds may be found in Mallinger et al. (Mallinger et al., 2015) (e.g. for CCT251545); Mallinger et al. (Mallinger et al., 2016) (e.g. for CCT251921 or Compound 42 / MSC2530818); Czodrowski et al., (Czodrowski et al., 2016)(e.g. for MSC2530818); Clarke et al. (Clarke et al., 2016)_(e.g. for CCT251921 or MSC2530818); all of which are herein incorporated by reference. CDK8 inhibitors are also described in Ettl et al. (Ettl et al., 2022), which is herein incorporated by reference.
-
-
-
-
-
- Active variants of Senexin C can be found in Zhang et al. (Zhang et al., 2022), which is herein incorporated by reference.
-
-
-
-
-
-
-
-
- The skilled person will recognise that functional derivatives or variants of these CDK8i molecules may be available for use in the invention.
- In another embodiment, the CDK8i is a nucleic acid, such as an inhibitory RNA molecule. The CDK8i may be an siRNA capable of inhibiting the expression of CDK8, such as inhibiting the transcription of the gene encoding CDK8 or inhibiting translation of mRNA encoding CDK8. The CDK8i, such as a siRNA, may bind the sequence encoding CDK8 or a complementary sequence thereof. The siRNA may be 20-30 nucleotides long. The siRNA may comprise or consist of RNA or may comprise or consist of nucleotide analogues. The CDK8i may specifically bind (i.e. hybridise with) a sequence comprising or consisting of SEQ ID NO: 2. The CDK8i may be any CDK8 siRNA described herein.
- In one embodiment, the CCNCi is a nucleic acid, such as an inhibitory RNA molecule. The CCNCi may be an siRNA capable of inhibiting the expression of CCNC, such as inhibiting the transcription of the gene encoding CCNCi or inhibiting translation of mRNA encoding CCNCi. The CCNCi, such as a siRNA, may bind the sequence encoding CCNC or a complementary sequence thereof. The siRNA may be 20-30 nucleotides long. The siRNA may comprise or consist of RNA or may comprise or consist of nucleotide analogues. The CCNCi may specifically bind (i.e. hybridise with) a sequence comprising or consisting of SEQ ID NO: 3 The CCNCi may be any CCNC siRNA described herein.
- It will generally be appreciated that the above inhibitors are specific examples of compounds that are known to inhibit CDC7 and/or CDK8. Derivatives and variants of these specific compounds are likely to work in a similar manner to these compounds, even if their activity is not optimal.
- Reference to a particular compound also includes variants where in one or more tertiary amine of the CDC7i and/or CDK8i (i.e. amines that are bonded to no hydrogen atoms) the N atom is replaced by P. Similarly, a reference to a particular CDC7i and/or CDK8i compound also includes variants where in one or more secondary and/or primary amine(s) of the compound (i.e. amines that are bonded to one or two hydrogen atoms) the N atom is replaced by O, S or P. Reference to a particular compound also includes variants where primary amines and phosphines are functionalised by one or two RZ groups, and/or where secondary amines and phosphines are functionalised by one RZ group.
- Reference to a particular compound also includes variants where alcohols and/or ethers present in the compounds are replaced by NH, NRZ, S, PH or PRZ.
- Reference to a particular compound also includes variants where halogens (e.g. F, Cl, Br and I) are replaced with another halogen (e.g. F, Cl, Br or I), H, CN, NH2, NHRZ NRz 2, PH2, PHRZ, PRZ 2, OH, ORz SH or SRZ. Reference to a particular compound also includes variants where a nitrile group is replaced with a halogen (e.g. F, Cl, Br or I), H, OH, NH2, NHRZ, NRZ 2, PH2, PHRZ, PRZ 2, ORz or SRZ.
- Reference to a particular compound also includes variants where one or more primary amide is substituted by one or two are functionalised by one or two RZ groups, and/or where secondary amides are functionalised by one RZ group.
- RZ represents alkyl, alkenyl and/or aryl group(s). RZ groups may be C1-8 alkyl, C2-8 alkenyl or C5-8 aryl group(s), for example C1-6 alkyl, C1-6 alkenyl or C5-6 aryl group(s), preferably C1-4 alkyl or C2-4 alkenyl group(s).
- Reference to a particular compound also includes variants where one or more (e.g. two or more) of the hydrogen atoms in the hydrocarbon chain of an alkyl, alkenyl and/or alkynyl groups are replaced with substituent groups. In one embodiment from 1 to 10 hydrogen atoms in the group are substituted, such as from 1 to 6, e.g. 1, 2, 3 or 4 of the hydrogen atoms in the hydrocarbon chain might be replaced with substituent groups. When more than one hydrogen atom in the group is replaced, the substituent groups used may be the same or may be different. For example, the alkyl, alkenyl or alkynyl group may be substituted with one or more substituent groups independently selected from halo (e.g. F, Cl, Br and I), hydroxyl, amino and carboxyl groups, and aryl or heteroaryl groups (especially unsaturated cyclic and heterocyclic groups with 5 to 10 atoms (e.g. 6 to 10 atoms) in their ring, such as imidazolyl, thiazolyl, thienyl, phenyl, tolyl, xylyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl or naphthyl groups). The alkyl, alkenyl or alkynyl group may be substituted with one or more substituent groups independently selected from halo (e.g. F, Cl and Br) hydroxyl and amino and carboxyl groups. The alkyl, alkenyl or alkynyl group may be substituted with one or more substituent groups independently selected from aryl or heteroaryl groups, especially unsaturated cyclic and heterocyclic groups with 5 to 10 atoms (e.g. 6 to 10 atoms) in their ring, such as imidazolyl, thiazolyl, thienyl, phenyl, tolyl, xylyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl or naphthyl groups. The ring itself may be substituted, e.g. with one or more C1-6 alkyl groups, such as one or two (or more) methyl or ethyl groups, as is the case in tolyl and xylyl. Preferably the total number of carbon atoms in each of the substituent groups is from 5 to 12.
- Reference to a particular compound also includes variants where the length of chains, such as alkyl and polyether (e.g. polyethylene glycol) chains are shortened or lengthened, for example by ten or fewer carbon atoms or polyether units, such as 6 or fewer, or 4 or fewer, or two (e.g. one) or fewer carbon atoms or polyether units. For example, a methyl group shown in a particular compound may be an ethyl group in a variant of the compound.
- Reference to a particular compound also includes variants that are ionic forms, salts, solvates, conformers, isomers, tautomers, N-oxides, esters, prodrugs, isotopes and protected forms thereof, for example, as discussed below; preferably, ionic forms, or salts or tautomers or isomers or N-oxides or solvates thereof; more preferably, ionic forms, or salts or tautomers or solvates or protected forms thereof; even more preferably the salts or tautomers or solvates thereof. Many compounds, especially those including acidic and/or basic moieties, can exist in the form of salts, for example acid addition salts or, in certain cases salts of organic and inorganic bases such as carboxylate, sulphonate and phosphate salts. All such salts are within the scope of this invention, and reference to a compound includes the salt forms of the compound.
- The salt forms of the compounds of the invention are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al. (Berge et al., 1977). However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compounds of the invention, also form part of the invention.
- The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in (Stahl et al., 2011; Wermuth and Stahl, 2002). Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used. The compounds of the invention may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed. Acid addition salts may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include salts formed with an acid selected from the group consisting of acetic, hydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulphonic, toluenesulphonic, methanesulphonic (mesylate), ethanesulphonic, naphthalenesulphonic, valeric, acetic, propanoic, butanoic, malonic, glucuronic and lactobionic acids. Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-Lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulphonic, sebacic, stearic, succinic and tartaric acids. If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO-), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth metal cations such as Ca2+ and Mg2+, and other cations such as Al3+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 +) and substituted ammonium ions (e.g., NH3R+, NH2R2 +, NHR3 +, NR4 +). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 +.
- Where the CDC7i and/or CDK8i compounds contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Reference to a CDC7i and/or CDK8i compound includes reference to such quaternary ammonium compounds. CDC7i and/or CDK8i compounds containing an amine function may also form N-oxides. A reference herein to a CDC7i and/or CDK8i compound) that contains an amine function also includes the N-oxide. Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle. N-Oxides can be formed as described in Advanced Organic Chemistry (Teesdale-Spittle, 1993) and by L. W. Deady (Deady, 1977).
- The compounds of the invention may form solvates, for example with water (i.e., hydrates) or common organic solvents. As used herein, the term "solvate" means a physical association of the compounds of the present invention with one or more solvent molecules. This physical association involves varying degrees of intermolecular, ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. The term "solvate" is intended to encompass both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include compounds of the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine. The compounds of the invention may exert their biological effects whilst they are in solution. Furthermore, the compounds of the present invention may have one or more polymorph (crystalline) or amorphous forms and as such are intended to be included in the scope of the invention.
- CDC7i and/or CDK8i compounds may exist in a number of different isomeric (e.g. geometrically isomeric or stereoisomeric), and tautomeric forms and references to those compounds include all such forms. For the avoidance of doubt, where a compound can exist in one of several geometric isomeric or tautomeric forms and only one is specifically described or shown, all others are nevertheless embraced the present invention. Other examples of tautomeric forms include, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/enediamines, nitroso/oxime, thioketone/enethiol, and nitro/aci-nitro.
- Where CDC7i and/or CDK8i compounds contain one or more chiral centres, and can exist in the form of two or more optical isomers, references to those compounds include all optical isomeric forms thereof (e.g. enantiomers, epimers and diastereoisomers), either as individual optical isomers, or mixtures (e.g. racemic mixtures) of two or more optical isomers, unless the context requires otherwise. Where CDC7i and/or CDK8i compounds exist as two or more optical isomeric forms, one isomer (e.g. enantiomer) in a pair of isomers may exhibit advantages over the other enantiomer, for example, in terms of biological activity. Thus, in certain circumstances, it may be desirable to use as a therapeutic agent only one of a pair of isomers (e.g. enantiomers), or only one of a plurality of diastereoisomers. Accordingly, the invention provides compositions containing a CDC7i and/or CDK8i compound having one or more chiral centres, wherein at least 55% (e.g. at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compound is present as a single optical isomer (e.g. enantiomer or diastereoisomer). In one embodiment, 99% or more (e.g. substantially all) of the total amount of the compound may be present as a single optical isomer (e.g. enantiomer or diastereoisomer).
- The compounds of the invention include compounds with one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element. For example, a reference to hydrogen includes within its scope 1H, 2H (D), and 3H (T). Similarly, references to carbon and oxygen include within their scope respectively 12C, 13C and 14C and 16O and 18O.
- Reference to CDC7i and/or CDK8i compounds may include reference to prodrugs thereof, for example esters such as carboxylic acid esters and acyloxy esters of the CDC7i and/or CDK8i compounds bearing a carboxylic acid group or a hydroxyl group. Esters are compounds containing the group -C(=O)OR, wherein R is an ester substituent, for example, a C1-6 alkyl group, a heterocyclyl group, or a C5-20 aryl group, preferably a C1-6 alkyl group. Particular examples of ester groups include methyl, ethyl, t-butyl and phenyl esters. Examples of acyloxy (reverse ester) groups are represented by -OC(=O)R, wherein R is an acyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Particular examples of acyloxy groups include -OC(=O)CH3 (acetoxy), -OC(=O)CH2CH3, -OC(=O)C(CH3)3, -OC(=O)Ph, and -OC(=O)CH2Ph. The term "prodrug" means any compound that is converted into a biologically active compound in vivo. For example, some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). The prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative. During metabolism, the ester group (-C(=O)OR), sugar derivative or other glycoside conjugate can be cleaved to yield the active drug.
- The subject may be mammalian, such as human. In one embodiment, the subject is a human patient afflicted with, or at risk of, cancer.
- The administration of the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be sequential, for example, immediately or within minutes, hours or days. Alternatively, the administration of the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be concurrent. The administration may be concurrent where the inhibitors are co-formulated.
- In one embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor are administered within one day of each other. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 48 hours of each other. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 35 hours of each other. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 30 hours of each other. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 24 hours of each other. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 16 hours of each other. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 12 hours of each other. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 8 hours of each other. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered within 6 hours of each other. Preferably the CDC7 inhibitor and CDK8 inhibitor are administered within 1 hour of each other.
- Where separate administration occurs, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 1 hour apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 4 hours apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 8 hours apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 12 hours apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 18 hours apart. In another embodiment, the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered at least 24 hours apart.
- The CDC7 inhibitor and CDK8 inhibitor may be administered between about 4 hours and about 48 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 8 hours and about 48 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 12 hours and about 48 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 4 hours and about 30 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 4 hours and about 24 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 8 hours and about 30 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 8 hours and about 24 hours apart. The B CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 12 hours and about 30 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 12 hours and about 24 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered between about 18 hours and about 30 hours apart. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be administered about 24 hours apart.
- Where separate administration occurs, the CDC7 inhibitor may be administered before the CDK8 (and/or CCNC) inhibitor. Alternatively, where separate administration occurs, the CDK8 (and/or CCNC) inhibitor may be administered before the CDC7 inhibitor.
- The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be provided in a composition together or provided in separate compositions. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may optionally be administered in combination with another therapeutically active agent. Other therapeutically active agents may comprise other immune adjuvants such as lymphokines and cytokines. Examples include interferons such as alpha, beta, and gamma interferon, interleukins such as IL-2, 11-4, IL-6, IL-13 et al., colony stimulating factors, TNFs, and the like. Other therapeutically active agents may comprise other antitumor agents such as chemotherapeutics and cytotoxins commonly used for treating cancer, agents that inhibit angiogenesis, and the like. These additional therapeutic agents may be administered separately or in combination. These additional therapeutic agents may be co-formulated with the CDC7 inhibitor and/or CDK8 (and/or CCNC) inhibitor.
- The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be administered locally or systemically by any method known in the art including but not limited to intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intranasal, oral or other mucosal routes. Additional routes include intracranial (for example intracisternal, or intraventricular), intraorbital, ophthalmic, intracapsular and intraspinal administration. It is envisaged that injections will be the primary route for therapeutic administration of the compositions although delivery through a catheter or other surgical tubing is also used. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal and intramuscular administration. Liquid formulations may be utilised after reconstitution from powder formulations.
- The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be administered in a suitable, nontoxic pharmaceutical carrier, or can be formulated in microcapsules or a sustained release implant. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be administered multiple times, if desired. The appropriate route, formulation, and immunization schedule can be determined by one skilled in the art.
- The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be administered with a physiologically acceptable carrier such as physiological saline. The composition may also include another carrier or excipient such as buffers, such as citrate, phosphate, acetate, and bicarbonate, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins such as serum albumin, ethylenediamine tetraacetic acid, sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerol and the like. The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor can be formulated in various ways, according to the corresponding route of administration.
- The administration may be a therapeutically effective amount.
- The CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor agent, or otherwise compositions of the invention, are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. The compositions of the invention are particularly relevant to the treatment of existing tumours, especially cancer, and in the prevention of the recurrence of such conditions after initial treatment or surgery. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980 (Remington and Osol, 1980).
- The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.
- The combination of the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may facilitate a significant increase in survival of treated mice in a colorectal cancer COLO205 xenograft model, or alternatively a breast cancer MDA-MB-231, MDA-MB-468 or U-2-OS model.
- The combination of the CDC7 inhibitor and CDK8 (and/or CCNC) inhibitor may be provided to patients/subjects that have failed to respond adequately to the standard care.
- In one embodiment, the cancer to be treated or prevented may comprise a solid tumour malignancy. The cancer cell may comprise or consist of a cancer cell of a solid tumour. The solid tumour may comprise a sarcoma, carcinoma, or lymphoma. The cancer may comprise neuroblastoma or melanoma. Solid tumors that can be treated using the compositions and methods described herein may be selected from any one of the group of solid tumors of the breast, lung, colon, stomach, liver, kidney, ovary, and prostate; or combinations thereof. Tumors that can be treated in accordance with the invention may be selected from breast carcinomas, lung carcinomas, prostate carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, vulval carcinomas, kidney carcinomas, cervical carcinomas, endometrial carcinoma, endometrial hyperplasia, endometriosis, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas, sarcomas include fibrosarcomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, brain tumors, renal cell carcinomas, abnormal vascular proliferation associated with phakomatoses, and edema (such as that associated with brain tumors); or combinations thereof. The cancer cell may comprise a cell of any one of the above solid tumours.
- Additionally or alternatively, the cancer to be treated or prevented may be a blood cancer, such as leukemia, lymphoma or myeloma.
- In one embodiment, the cancer to be treated or prevented is osteosarcoma or epithelial adenocarcinoma.
- According to another aspect of the invention, there is provided a CDC7 inhibitor in combination with a CDK8 and/or CCNC inhibitor for use in the treatment or prevention of cancer in a subject.
- According to another aspect of the invention, there is provided a composition comprising a CDC7 inhibitor and a CDK8 and/or CCNC inhibitor.
- According to another aspect of the invention, there is provided a kit for treatment or prevention of cancer in a subject, the kit comprising:
- i) a CDC7 inhibitor; and
- ii) a CDK8 and/or CCNC inhibitor.
- According to another aspect of the invention, there is provided a composition or kit according to the invention for use as a medicament.
- According to another aspect of the invention, there is provided a composition according to the invention, or kit according to the invention, for use in the treatment or prevention of cancer in a subject.
- According to another aspect of the invention there is provided a method of treatment or prevention of cancer in a subject, wherein the cancer is determined or known to be deficient in functional CDK8 and/or CCNC expression, the method comprising the administration of a Cell Division Cycle 7 kinase inhibitor (CDC7i) to the subject.
- According to another aspect of the invention there is provided a Cell Division Cycle 7 kinase inhibitor (CDC7i) for use in a method of treatment or prevention of cancer in a subject, wherein the cancer is determined or known to be deficient in functional CDK8 and/or CNCC expression.
- The invention recognises that some subjects may have a cancer that is CDK8 deficient, which would then obviate the need for a CDK8i and/or CCNCi to be administered in combination with a CDC7i. The skilled person will recognise that cancers deficient in CDK8 and/or CNCC represent a sub-class of cancers that may be targeted for treatment more efficiently by single CDC7i therapy.
- For example, a percentage of Diffuse large B cell lymphoma (DLBCL) is understood to be CDK8 deficient. Therefore, the method may comprise the treatment of DLBCL having CDK8 deficiency with a CDC7 inhibitor (CDC7i).
- CDK8 deficiency has been implicated in some cases of Sarcoma (1.6 %; 4/255 cases); Lung Adenocarcinoma (1.1 % (6/566 cases); and Diffuse Large B-Cell Lymphoma (1.2 %; 1/48 cases). Therefore, the method may comprise the treatment of such cancers having CDK8 deficiency with a CDC7 inhibitor (CDC7i).
- CCNC deficiency has been implicated in some cases of Diffuse Large B-Cell Lymphomas (10.42 %; 5/48 cases); Prostate Adenocarcinoma (8.7 %; 43/494 cases), Uveal Melanoma (6.3 %; 5/80 cases); Liver Hepatocellular Carcinoma (2.4 %; 9/372 cases); Stomach Adenocarcinoma (1.6 %; 7/440 cases); and Bladder Urothelial Carcinoma (1.5 %; 6/411 cases). Therefore, the method may comprise the treatment of such cancers having CCNC deficiency with a CDC7 inhibitor (CDC7i).
- CDK8 deficiency is understood to mean that the cells, such as the cancer cells, do not express functional CDK8, or the CDK8 expression is significantly lower than average expression levels in normal cells (i.e. non-CDK8-deficient cells), such as at least 3-fold, 5-fold, 10-fold, 50-fold or 100-fold lower.
- CCNC deficiency is understood to mean that the cells, such as the cancer cells, do not express functional CCNC, or the CCNC expression is significantly lower than average expression levels in normal cells (i.e. non- CCNC -deficient cells), such as at least 3-fold, 5-fold, 10-fold, 50-fold or 100-fold lower.
- The invention has advantageously found that tumors defective in either CDK8 and/or CCNC can be particularly sensitive to CDC7i-basesd chemotherapy.
- Therefore, according to another aspect of the invention, there is provided the use of CDK8 and/or CCNC as biomarkers for predicting the efficacy of CDC7i therapy for cancer of a subject, wherein the reduction or absence of expression of functional CDK8 and/or CCNC in a subject is indicative of an increased efficacy of the CDC7i therapy for cancer.
- According to another aspect of the invention, there is provided a method of predicting the efficacy of a CDC7i therapy for cancer in a subject, the method comprising determining the levels of expression of functional CDK8 and/or CCNC in the subject, or obtaining results of such a determination,
wherein a decreased level of expression of CDK8 and/or CCNC in the subject is indicative of a more effective CDC7i therapy for the cancer in the subject. - According to another aspect of the invention, there is provided a method of selecting a subject for treatment for cancer with a CDC7i, the method comprising determining the levels of expression of functional CDK8 and/or CCNC in the subject, or obtaining results of such a determination,
wherein the subject is selected for CDC7i treatment if they have a decreased level of expression of CDK8 and/or CCNC. - The methods or uses of the invention may further comprise the treatment of the subject with a CDC7i, for example by administration of a CDC7i to the subject.
- According to another aspect of the invention, there is provided a method of treatment of a subject for cancer, the method comprising:
- determining the levels of expression of functional CDK8 and/or CCNC in the subject, or
- having obtained results of the levels of expression of functional CDK8 and/or CCNC in the subject; and
- administering CDC7i treatment if the subject has a decreased level of expression of CDK8 and/or CCNC.
- The increased efficacy may be relative to cancer subjects having normal or higher levels of expression of functional CDK8 and/or CCNC. The increased efficacy may be a statistically significant increase in efficacy. The decreased level of expression of CDK8 and/or CCNC may be relative to a reference level. The reference level may be an average level of expression in a general population, or an average level in a population of subjects having cancer, preferably the same cancer type and/or stage.
- The reduction or absence of expression of functional CDK8 and/or CCNC in a subject may be in a selected tissue, such as tumour tissue or cells. For example the tumour tissue or cells of the subject may be determined for their expression of functional CDK8 and/or CCNC.
- The reduction/decreased level of expression of functional CDK8 and/or CCNC may be a 100% decrease. In another embodiment, the decreased level of expression of CDK8 and/or CCNC may be a 50%, 80%, 90%, 95% or 99% decrease.
- The CDC7i may be administered and/or formulated as described herein. The CDC7i may be a The CDC7i described herein.
- The term "inhibitor" is understood to refer to an agent that is capable of preventing or reducing the activity of a target molecule, or a biological pathway. The inhibition may be complete, i.e. 100% inhibition, or the inhibition may be partial. For example, the inhibition may inhibit up to 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the original activity of the target (i.e. relative to the activity when not in the presence of the inhibitor). The inhibition may be sufficient to effect a statistically significant reduction in activity of the target. In one embodiment, the level of inhibition is sufficient to cause a therapeutic effect.
- The term "subject" means all animals including humans. Examples of subjects include humans, cows, dogs, cats, goats, sheep, and pigs. The term "patient" means a subject having a disorder in need of treatment.
- A 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
- The term "functional" is understood to refer to the normal function of a given biological molecule such as CDC7, CDK8 or CCNC. A loss of function may be complete, such that it cannot carry out its normal ligand binding and/or reaction, or it may have a significant impairment in activity, such as a lower ability to bind its ligand or a lower rate of reaction. A loss of "function" or "non-functional molecule" may be a loss of the activity of the molecule, or a lack of expression of the molecule.
- The term "functional expression" is understood to refer to the expression of a functional molecule having its inherent activity.
- The skilled man will appreciate that preferred features of any one embodiment and/or aspect of the invention may be applied to all other embodiments and/or aspects of the invention.
- Examples embodying an aspect of the invention will now be described with reference to the following figures:
-
Figure 1 : Genome-wide CRISPR/Cas9 screen identifies genes that modulate proliferation in response to a CDC7 inhibitor- (a) Representation of the genome-wide CRISP/Cas9 screen workflow used to identify genes, which upon gene editing, either enhance or suppress the anti-proliferative effects of XL413 indicated by grey dot-dash or black broken circles, respectively.
- (b) Ranked plot of the differential between the XL413-treated and control beta scores obtained from MAGeCKFlute analysis. Significant gene outliers were identified using thresholds of +/- 2 standard deviations from the mean differential. Lighter grey dots represent candidate genes, which upon gene editing, either significantly enhance (179 genes) or suppress (96 genes) the anti-proliferative effects of XL413, respectively. Genes of interest have been highlighted and named.
-
Figure 2 : CDK8 and CDC7 inhibitors synergise to restrict cell proliferation and DNA synthesis- (a and b) MCF10A cells were plated at equal low density and then either mock treated or treated with 31 nM BI-1347, in the presence or absence of XL413 at a doses of (a) 1.25 µM or (b) 10 µM over a 7 day period. Representative images of crystal violet-stained cells are shown alongside quantitation of crystal violet intensity normalised to the untreated control. Graph shows the mean ± SD of three independent experiments.
- (c) A typical profile obtained by flow cytometry of MCF10A cells labelled with EdU and DAPI that indicates the gating applied in order to determine the percentage of cells with sub-G1 DNA content (sub-G1), G1 cells (G1), replicating early-mid S phase cells (Early-Mid S / EdU+), replicating mid-late S phase cells (Mid-Late S / EdU+), cells with S phase DNA content but not replicating (2n< & <4n / EdU-), and cells either in G2 or mitosis (G2/M).(d-f) MCF10A cells were mock treated or treated with 10 µM XL413, in the presence or absence of increasing concentrations of the CDK8 inhibitor BI-1347, for 24 hours. (d) Cells were labelled with EdU for last 30 minutes and DNA synthesis, DNA content and cell cycle distribution were assessed by flow cytometry and analysed using the gating strategy described in above (
Figure 2c ). (e) Distribution of cells in the cell cycle. (f) Fluorescence intensity, proportional to EdU incorporation, in individual S phase cells. Long and short horizontal black lines indicate the median and the interquartile range, respectively, extending from the 25th to the 75th percentile for ~3,810 cells for each condition. Data is from a single experiment.
-
Figure 3 : CDK8 activity is required for efficient DNA synthesis under conditions of limited CDC7 kinase activity (a - c) MCF10A cells were mock treated or treated with 31 nM BI-1347, in the presence or absence of increasing concentrations of the CDC7 inhibitors, XL413 (a-c) and TAK-931 (d-f), for 24 hours. (a and d) Cells were labelled with EdU for last 30 minutes and DNA synthesis, DNA content and cell cycle distribution were assessed by flow cytometry and analysed using the gating strategy described inFigure 2c .c (b and e) distribution of cells in the cell cycle. (c and f) Fluorescence intensity, proportional to EdU incorporation, in individual S phase cells. Long and short horizontal black lines indicate the median and the interquartile range, respectively, extending from the 25th to the 75th percentile for ~4,190 cells for each condition. Data is from a single experiment. - (a - c) Proliferation curves of MCF10A EditR cells expressing sgRNAs from CRISPR library modules I, II and III in the absence (untreated) or presence of 1.25 µM XL413. Data is from three independent counts of each sample ± standard deviation (SD). (d and e) DNA extracted from samples of the CRISPR/Cas9 screen, which were harvested on
day 0 and after 20 or 24 days of growth in the absence (untreated) or presence of 1.25 µM XL413, respectively, was analysed by next generation sequencing. Sequence data was mapped to a CELLECTA genome-wide sgRNA reference library using the MAGeCKFlute pipeline. (d) The table summarises the total and missing number of sgRNAs, the calculated % sgRNA coverage and the number of missing genes. - (a - e) Dot plots of normalised sgRNA read counts for
day 0,day 20 untreated andday 24 XL413-treated samples for genes that express the core components of the CDK8/CDK19-kinase module of the mediator complex. Dots are connected by lines as a visual aid to track abundance changes of sgRNAs across different samples. (f) Protein sequences for CDK8 (NP001251, amino acids: 1 to 464) and CDK19 (NP055891, amino acids: 1-502) were aligned using Clustal Omega, showing 384 identical sites with an overall pairwise identity of 75 %. The degree of conservation of aligned amino acid residues in each column is summarised as a bar graph. - A panel of different cell lines, including: MDA-MB-468 (a - c); MDA-MB-231 (d - f); and U-2 OS (g - i), were mock treated or treated with 1.25 µM XL413 and/or 31 nM BI-1347BI-1347 for 24 hours. Cells were labelled with EdU for last 30 minutes and DNA synthesis, DNA content and cell cycle distribution were assessed by flow cytometry and analysed using the gating strategy described in
Figure 2c . (b, e and h) distribution of cells in the cell cycle. (c, f and i) Fluorescence intensity, proportional to EdU incorporation, in individual S phase cells. Long and short horizontal black lines indicate the median and the interquartile range, respectively, extending from the 25th to the 75th percentile for ~ 3,922 (MDA-MB-468), ~ 3,291 (MDA-MB-231), and ~4,628 (U-2 OS) cells for each condition. Data is from a representative experiment from 3 biological repeats. - Eukaryotic DNA replication initiates at multiple origins according to a well-defined spatiotemporal program, with origins being activated in early, mid-, or late S phase, each one giving rise to two replication forks (Gilbert et al., 2010). Origin activation is orchestrated by the coordinate action of Cyclin-dependent kinases (CDKs) and CDC7 kinase, also known as DDK (DBF4-dependent kinase). In this endeavour CDC7 is known, by phosphorylating several subunits of the MCM complex (Gilbert et al., 2010; Masai et al., 2006; Sheu and Stillman, 2006, 2010), to support formation and activation of the Cdc45-MCM-GINS (CMG) helicase complex (Labib, 2010).
- Many small-molecule ATP-competitive CDC7 inhibitors (CDC7is) have been developed as the focus of multiple drug discovery programs in oncology as an alternative strategy to restrain DNA replication by limiting origin firing instead of directly blocking fork progression (Montagnoli et al., 2010). Inhibition of CDC7 in cells leads mitotic abnormalities as well as replication stress, with decreased origin activation, delayed S-phase progression and activation of the S-phase checkpoint (Iwai et al., 2019; Rainey et al., 2020). The latter involves ETAA1-dependent activation of ATR to further suppress origin activation and prevent premature entry and catastrophic progression through an aberrant mitosis with under-replicated DNA (Rainey et al., 2020).
- Several CDC7is have shown potent anti-proliferative activity in vitro, and in several cases, excellent anti-tumor activity in preclinical models in vivo (Iwai et al., 2019, 2021; Montagnoli et al., 2008), with the most advanced compounds currently in early open clinical trials, including: LY3143921 (Clinicaltrials.gov ID: NCT03096054), and TQB3824 (NCT05028218), or in trials that have been completed or terminated: BMS-863233 (NCT00838890 and NCT00886782), NMS-1116354 (NCT01016327 and NCT01092052) and TAK-931 (NCT02699749).
- To better guide the clinical development a recent study combined the CDC7i, TAK-931 with different chemotherapeutic drugs, and showed that the combination of TAK-931 with several DNA-damaging agents synergized to produce the strongest anti-proliferative effects in vitro. The efficacy of combination therapies was confirmed using preclinical primary-derived xenograft models (Iwai et al., 2021). However, in order to properly position CDC7 inhibitors in the clinic we require (1) a better understand of how tumour cells respond to this new class of drugs and (2) how to increase their efficacy without affecting their tolerability so that the therapeutic window is improved.
- While CDC7 is an essential gene in almost every cell line tested thus far (Cowley et al., 2014; McFarland et al., 2018; Meyers et al., 2017; Rainey et al., 2017; Tsherniak et al., 2017), it is now clear that different cancer cells have a wide range of sensitivity to either partial or transient CDC7 inhibition (Iwai et al., 2019; McDonald et al., 2017; Sasi et al., 2014). This suggests that multiple factors may determine how cells respond to decreased levels of CDC7 activity and that the response to limited origin firing is genetically controlled.
- To understand how the problems in DNA replication arising from CDC7 inhibition are dealt with we performed a genome-wide CRISPR/Cas9 chemogenetic screen to identify genes, that upon loss, could sensitize to the anti-proliferative effects of CDC7i. As our top hit we identified CDK8 and its cognate Cyclin, Cyclin C (CCNC), as a significant hit.
- CDK8 is a ubiquitously expressed nuclear Ser/Thr kinases, that is a component of the "Mediator-CDK8 module", and is traditionally classified as a "transcriptional" rather than a "cell cycle" CDK (Dannappel et al., 2019; Galbraith et al., 2010). The Mediator is a large multi-subunit complex, consisting of a "head", "middle", and "tail" at the core of its structure, which is essential for its function in transcription by bridging transcriptional elements, promoters, chromatin modifiers and transcription factors (TFs) to RNA polymerase II (Fant and Taatjes, 2019; Harper and Taatjes, 2018). The "CDK8 module" is a 4 subunit regulatory subcomplex in which Cyclin C (CCNC) and MED12, activate CDK8 kinase function, and MED13, enables their interaction with the Mediator (Dannappel et al., 2019; Fant and Taatjes, 2019; Menzl et al., 2019). In mammals a paralog of CDK8, namely CDK19, exists and has high sequence similarity to CDK8. CDK19 in a mutually exclusive manner with CDK8, can form an analogous "CDK19 module" (Tsutsui et al., 2008). To further complicate matters paralogs of MED12 and MED13 also exists, termed MED12L (MED12-like) and MED13L (MED13-like) respectively.
- CDK8, primarily regulates cellular function through phosphorylation of a large number of proteins including: Mediator components, TFs; chromatin remodelers, and RNA polymerase II, in addition, to non-mediator associated proteins (Poss et al., 2016). CDK8 activity is not required for bulk transcription, but it regulates the expression of subsets of genes in cell type- and context-specific manner. This uniquely highlights CDK8 as mediators of transcriptional reprogramming, rather than regulators of global transcription, like CDK7 and CDK9 (Chen et al., 2019; Fant and Taatjes, 2019; Nemet et al., 2014).
- CDK8 was first described as a proto-oncogene in colorectal cancer (CRC), where high expression levels negatively correlated with poor patient survival and were associated with advance stage disease progression (Firestein et al., 2008, 2010; Seo et al., 2010). Interestingly, downregulation of CDK8 reduced proliferation of CRC cells in vitro and in xenograft models with CDK8 amplification, highlighting the therapeutic potential of targeting CDK8 (Adler et al., 2012; Firestein et al., 2008; Seo et al., 2010). As a result several CDK8 inhibitors have now been developed as targeted agents and because of the similarity between CDK8 and CDK19 active site most of these compounds can be considered as dual CDK8/19 inhibitors (Fant and Taatjes, 2019; Philip et al., 2018; Xi et al., 2019). Some of these have shown cancer type-specific therapeutic effects in in vivo colorectal (Liang et al., 2018), breast (McDermott et al., 2017), leukemia (Pelish et al., 2015; Rzymski et al., 2017), prostate (Nakamura et al., 2018) and lung (Porter et al., 2012) models. Inhibition of CDK8 signalling was also shown to modulate the cytotoxic effects of NK cells, promoting tumour surveillance and increasing survival in mouse melanoma, leukemia and metastasising breast cancer models (Hofmann et al., 2020; Putz et al., 2013).
- Taken together all these observations indicate that the antitumor activity of CDK8 inhibitors could be mediated by both cell-intrinsic mechanisms, leading to reduced tumour cell proliferation and survival, as well as cell-extrinsic mechanism mediated by the host immune system and tumor microenvironment.
- Thus, CDK8 inhibition is a potentially promising therapeutic approach, however, as in the case of CDC7is, the development of CDK8 based therapy has obstacles to overcome. For example tolerability issues have been observed with some inhibitors (Chen et al., 2019) and as a result only select inhibitors, RVU120 and BCD-115, have progressed to clinical trials targeting acute myeloid leukemia (ClinicalTrials.gov Identifier: NCT04021368) and estrogen-receptor positive breast cancers (ClinicalTrials.gov Identifier: NCT03065010), respectively. Due to the cell-type specific regulation of gene expression by CDK8, the best indication for a CDK8i based therapy has not yet been identified and maybe limited to only a few cancer types/subtypes. Finally, all CDK8 inhibitors identified so far also target CDK19, and developing monospecific CDK8 inhibitors could be a challenge due to the high sequence similarity shared between CDK8 and CDK19 (Philip et al., 2018; Tsutsui et al., 2008).
- While described as transcriptional mediators, a recent paper has shown that CDK8/CDK19-CCNC can bind to the human homolog of yeast sld7 protein, MTBP, an essential DNA replication origin firing factor (Boos et al., 2013). This association occurs independently from MED12 and MED13, suggesting this could represent a role independent from the Mediator complex and transcription (Köhler et al., 2019). This interaction is mediated by a metazoan specific binding region in MTBP that is important for efficient DNA synthesis, preventing replication stress and ensuring complete genome duplication to support a normal mitosis (Köhler et al., 2019). However, while CDK8/CDK19-CCNC can bind and phosphorylate MTBP in vitro, CDK8 dependent phosphorylation of MTBP in living cells has yet not been detected nor studied (Ferreira et al., 2021; Köhler et al., 2019).
- In this work we show that a dual-specificity inhibitor of CDK8/CDK19 only very subtly reduces the rate of DNA synthesis, but in combination with the CDC7 inhibitors, XL413 and TAK-931, dramatically blocks DNA synthesis and cell proliferation in a variety of human cell lines. We propose that inhibition of CDK8 can strongly potentiates the broad antitumor activity of CDC7 inhibition and, by doing so, it can increase the spectrum of tumour responding to a CDK8i and CDC7i combination therapy compared to monotherapy.
- In order to identify genes that change the rate of proliferation when DNA synthesis is mildly limited by CDC7 inhibition, we performed a CRISPR/Cas9 genome-wide knockout screen as outlined in
Figure 1a . Briefly, MCF10A EditR cells that stably express Cas9 were transduced with a lentiviral-sgRNA library targeting approximately 19,000 coding genes and then were either untreated or treated with 1.25 µM of selective ATP competitive CDC7 inhibitor, XL413 (Koltun et al., 2012). Cell proliferation was monitored over the course of the screen and, consistent with previous findings (Rainey et al., 2017), this concentration of XL413 only mildly suppressed cell proliferation in this cell line (Supplemental Figure 1a-c ). Next generation sequencing was then used to determine sgRNA abundance atday 0,day 20 untreated andday 24 XL413 treated cells. Preliminary analysis showed that the sgRNA library representation at the beginning of the experiment and at the end-points was nearly complete and that full coverage of all protein encoding genes was achieved in the screen (SupplementalFigure 1d ). CRISPR/Cas9-mediated loss of function of genes involved in supporting proliferation in untreated cells or in response to CDC7 inhibition is expected to result in a decreased relative abundance of the corresponding sgRNAs in the cell population. By analysing the data from the two experimental branches we identified ~179 genes that, when knocked out, potentially decreased the proliferation rate specifically in CDC7i treated cells (Figure 1b ). When the genes were ranked according to the magnitude of the effect, CDK8, a gene that expresses a component of the CDK8/CDK19-CCNC-MED 12-MED 13 mediator complex kinase-module (Dannappel et al., 2019; Fant and Taatjes, 2019; Menzl et al., 2019), came out as the top hit (Figure 1b and Table 1, ranked 19012 out of 19012 genes).Table 1. The table summarises the beta scores, assigned by MAGeCK-MLE, and based on changes of sgRNA abundance from day 0 today Rank Gene XL413 beta score Untreated beta score Differential beta score Uniprot ID Uniprot protein name and functional annotation 1 (∗1) RIF1 - 0.0162 -0.7603 0.7442 Q5UIP0 Replication timing regulatory factor 1; Telomere-associated protein RIF1; Key regulator of TP53BP1 that plays a key role in the repair of double-strand DNA breaks (DSBs) in response to DNA damage: acts by promoting non-homologous end joining (NHEJ)-mediated repair of DSBs.6089 MED12 0.8276 -0.9204 0.0928 Q93074 Mediator of RNA polymerase II transcription subunit 12; Component of the Mediator complex, a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Mediator functions as a bridge to convey information from gene-specific regulatory proteins to the basal RNA polymerase II transcription machinery. Mediator is recruited to promoters by direct interactions with regulatory proteins and serves as a scaffold for the assembly of a functional preinitiation complex with RNA polymerase II and the general transcription factors.10046 MED13 0.0939 0.0877 0.0062 Q9UHV7 Mediator of RNA polymerase II transcription subunit 13; Component of the Mediator complex, a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. NB: Mediator function and recruitment as described for MED12. 10184 CDK19 - 0.2393 -0.2427 0.0034 Q9BWU1 Cyclin- dependent kinase 8/11; Cyclin dependent kinase 19; Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family.18866(∗2) CCNC - 1.0759 -0.7157 -0.3602 P24862 genes. recruitment Cyclin-C; Component of the Mediator complex, a coactivator involved in regulated gene transcription of nearly all RNA polymerase II-dependent genes.
NB: Mediator function and recruitment as described for MED12.19012(∗2) CDK8 - 1.6094 -0.1703 -1.4391 P49336 Cyclin- dependent kinase 8; Component of the Mediator complex, a coactivator involved in regulated gene transcription of nearly all RNA polymerase II-dependent genes. Phosphorylates the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNAp II), which may inhibit the formation of a transcription initiation complex.NB: Mediator function and recruitment as described for MED12. - Significantly, CCNC, the cognate cyclin of CDK8, was also required to support efficient proliferation specifically in response to CDC7i, while the CDK8 paralog, CDK19, and other components of the mediator complex kinase-module, MED12 and MED13, were not required. In addition, we also identified ~96 genes, which upon loss of function, could increase the rate of proliferation specifically in cells treated with low dose CDC7i. Importantly RIF1, a gene we have showed could reduce the requirement for CDC7 kinase when deleted in our previous CRISPR/Cas9 screen study (Rainey et al., 2020), was once again ranked as the top hit in this gene cohort and instils confidence in the results of this screen. (
Figure 1b and Table 1, ranked 1 out of 19012 genes). - Interestingly CRISPR targeting of CDK8 showed significant and robust decreases in accumulation of multiple sgRNAs, but only under conditions of limited CDC7 activity (Supplemental
Figure 2a ). In contrast the targeting of the CDK8 paralog, CDK19, which shows 76% sequence identity (SupplementalFigure 2f ), and which is also regulated by CCNC, does not strongly or reproducibly affect sgRNA abundance between the untreated and CDC7i treated branches (SupplementalFigure 2b ). A similar lack of robust and reproducible changes in sgRNA abundance in response to CDC7i is also observed when MED12 and MED13 are targeted, which are required to facilitate the interaction between CDK8-CCNC and the transcriptional mediator complex (Dannappel et al., 2019; Fant and Taatjes, 2019; Menzl et al., 2019). - Taken together these observations suggest that loss of function of CDK8, and not its closely related paralog CDK19, sensitises cells to CDC7 inhibition, and that a role for CDK8 in supporting proliferation of cells with limited CDC7 kinase activity may be independent from its interaction and transcriptional role with the mediator complex.
- Recently, CDK8/CDK19-CCNC complex was shown to bind and phosphorylate the human replication initiation factor, MTBP, in vitro (Ferreira et al., 2021; Köhler et al., 2019). While the region of MTBP, required for binding CDK8/CDK19-CCNC complex, is important for normal genome replication, the requirement and role of CDK8/CDK19-CCNC in this process is poorly understood (Ferreira et al., 2021; Köhler et al., 2019). For this reason, we focused our analysis on determining their role in the response to CDC7 inhibition.
- In order to independently demonstrate that CDK8 activity allows cells to grow better when CDC7 is inhibited, we performed a long-term proliferation assays (
Figure 2a and b). Briefly, MCF10A cells were mock-treated or treated in the continued presence of a dual-specificity CDK8/CDK19 inhibitor, BI-1347, in the presence or absence of XL413 at two different concentrations (1.25µM and 10 µM) for a 7-day period. Relative cell proliferation was estimated by staining cells with crystal violet and measuring staining intensities. Compared to controls, the inhibition of CDK8/CDK19 with 31 nM BI-1347 alone resulted in a decrease in the relative staining intensity of approximately 30 % (Figure 2a and b ). Inhibition of CDC7, with either 1.25 µM or 10 µM of XL413 alone resulted in a relative intensity reduction of ~ 15 % and 70 % respectively. Importantly the addition of BI-1347 to XL413 caused a further reduction in cell number with staining intensity decreasing by 70 % and 95 % respectively. These results strengthen the idea that inhibition of CDK8 activity confers a growth disadvantage under conditions of limited CDC7 activity. - We next sought to understand if the effects of CDK8 and CDC7 inhibition on proliferation were related to altered dynamics of the DNA replication. For these experiments MCF10A cells were either mock-treated or treated with XL413 for 24 hours in the presence or absence of BI-1347 at increasing concentrations. Flow cytometry was used to determine the consequences on cell cycle distribution and the rate of DNA synthesis (
Figure 2c-f ). As previously shown XL413 treatment strongly reduced the rate of DNA synthesis (Alver et al., 2017; Hiraga et al., 2017; Rainey et al., 2017, 2020), leading to a characteristic accumulation of cells in mid to late S phase (Figure 2c-e ). Instead CDK8/CDK19 inhibition resulted in a dose-dependent increase in G1 phase cells with a corresponding decrease in early-mid S phase cells. Utilising a more refined analysis however we noticed a subtle and dose-independent decrease in the rate of DNA synthesis in S phase cells (Figure 2f ). Strikingly, in XL413 treated cells inhibition of CDK8/CDK19 resulted in a further accumulation of mid-late S phase cells with a profound and dose independent decrease in the rate of DNA synthesis (Figure 2c-f ). Together these results indicate CDK8/CDK19 activity is required to promote efficient DNA synthesis and such role in S-phase becomes critical when CDC7 activity becomes rate limiting. - By adopting the same experimental approach, we aimed to understand whether CDK8/CDK19 inhibition could sensitise cells CDC7 inhibitors across a wide range of concentrations. For these experiments and in order to rule out possible off-target effects we used two distinct CDC7 inhibitors, XL413 and TAK- 931 (Iwai et al., 2019). When cells were treated with either CDC7 inhibitor, in the absence of CDK8/CDK19i, we observed a dose-dependent accumulation of mid-late S phase cells and corresponding decrease in the rate of DNA synthesis of S phase cells (
Figure 3a-f ). Inhibition of CDK8/CDK19 alone had minimal impact on the cell cycle distributions and resulted in a slight decrease in rate of DNA synthesis of S phase cells. Strikingly, when together with the CDK8/CDK19 inhibitor both CDC7 inhibitors cased a more pronounced accumulation of cells in mid-late S phase of the cell cycle and much more profound decrease in rate of DNA synthesis at all doses tested. - Finally, to understand whether the decrease of DNA synthesis by CDK18/19 and CDC7 inhibitors is a general mechanism or is related to a nuance of MCF10A cells similar experiments were performed in a panel of cancer cell lines (MDA-MB-468, MDA-MB-231 and U2-O-S). In all these cell lines we observed a strong potentiation of cell cycle and DNA replication phenotypes imposed by CDC7 inhibition by the CDK8/CDK19 inhibitor BI-1347 (Supplemental
Figure 3 ). - Taken together these results indicate that inhibition of CDK8/CDK19 can potently sensitise cells to the effects of CDC7 inhibition on DNA replication dynamics, and that CDK8/CDK19 activity can modulate cellular responses to CDC7 inhibition in multiple cell types.
- Successful drug development requires the understanding of which patent population is most likely to benefit of any given new drug as well as whether a new drug can be administered as single agents or in a combination therapy. Such understanding for CDC7 inhibitors is still very limited and mostly based on empirical testing in preclinical models.
- We have embarked in finding the genes that can alter the response to CDC7 inhibitors using functional genomics approaches (Rainey et al., 2020). The genome wide-CRISPR/Cas9 screen presented in this work now indicates that loss of function of CDK8 and CCNC sensitizes cells to the anti-proliferative activity of CDC7 inhibition. This suggests that tumors defective in either CDK8 and/or CCNC could be particularly sensitive to CDC7i-basesd chemotherapy and that CDK8 and CCNC could be then developed as novel biomarkers of response. In a preliminary analysis of publicly available data form 10,967 cancer samples, form the cBioPortal TCGA PanCancer Atlas of 32 studies, we noted that CDK8 deletion is present in sarcomas (~4.31 % of 255 cases), lung adenocarcinomas (~2.47 % of 566 cases), and diffuse large B-cell lymphoma (~2.08 % of 48 cases). CCNC deletions instead is observed at high rate in diffuse large B-cell lymphoma (~12.5 % of 48 cases) prostate adenocarcinoma (~9.11 % of 494 cases) uveal melanoma (~7.5 % of 80 cases), liver hepatocellular carcinoma (~3.23 % of 372 cases) and stomach adenocarcinoma (~2.95 % of 440 cases), that show frequent deep of CCNC.
- We have then shown that inhibition of CDK8/CDK19 kinases by BI-1347 potently sensitizes cells to the anti-proliferative effects of CDC7 inhibition. Mechanistically we have shown that, while per se CDK8/CDK19 have a minor contribution to the rate of DNA synthesis, their inhibition in combination with even the lowest dose of CDC7 inhibitors tested, elicits a profound decrease in the rate of DNA synthesis, that in our replication assays appears to be equivalent to a ~10-fold increase in the potency of the CDC7 inhibitor.
- It is very likely that the synergistic decrease in proliferation and rate of DNA synthesis, observed upon combined CDK8/CDK19i and CDC7i, are not related to the role of CDK8 in transcription, but possibly to a direct role of CDK8 in DNA replication. In favour of this hypothesis is the fact that, while CDK8 was the top hit, neither MED12 nor MED13, which target CDK8 to the mediator complex for regulation transcription, nor the CDK19 paralogue, scored in our CRISPR screen.
- Such more fundamental role of CDK8 in DNA replication also strongly suggests that a CDC7i + CDK8i combination will be effective in blocking cell proliferation in a very large variety of cancers, thus very much expanding the scope of CDK8 inhibition which at present is limited to a small subset of cancers that have strong dependencies on CDK8 mediated gene expression.
- Finally we note that a recent study reported that siRNA mediated down regulation of CDK19 but not of CDK8 can prevent apoptosis induced by a dual specificity CDC7/CDK9 inhibitor, PHA767491, in several cancer cell lines (Sasi et al., 2017). Apoptosis induced by PHA-767491 maybe be driven by loss of anti-apoptotic proteins such as MCL1 (Natoni et al., 2011, 2013), among other gene expression changes, as a result of inhibition of CDK9. It plausible that CDK19 contributes to this apoptotic transcriptional response by regulating the expression of additional apoptotic genes. Such results are thus only apparently different from ours which instead indicate that only CDK8 is relevant to enhance proliferation when CDC7 is inhibited by XL413 and TAK931, which do not affect CDK9 activity.
- In summary, we identify CDK8 and CCNC are potential novel biomarkers whose functional status can modulate the anti-cancer effects of CDC7i monotherapy. Furthermore we show, for the first time, that inhibition of CDK8/CDK19 potentiates the anti-proliferative activity a of CDC7 inhibition suggesting that a combination of CDC7 with CDK8 inhibitors may improve efficacy and can be potentially efficacious across a broader spectrum of cancers when compared to either monotherapy.
- Cell culture was performed in a Class II Bio-safety cabinet and all cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cell count and viability were determined using a LunaII automated cell counter (Logos Biosystems) and trypan blue exclusion. Culture media and reagents were from Sigma-Aldrich unless stated otherwise. MCF10A cell line (human, female origin) was purchased from ATCC and used without further authentication. MCF10A-EditR cells (stably expressing Cas9) were generated as previously described (Rainey et al., 2017). MCF10A cells and derivatives were cultured using DMEM supplemented with 5% horse serum, 25 ng/ml cholera toxin, 10 µg/ml insulin, 20 ng/ml epidermal growth factor (Peprotech), 500 ng/ml hydrocortisone, 50 U/ml penicillin and 50 µg/ml streptomycin. Lenti-X™ 293T cell line (human, female origin) were purchased from TaKaRa and used without further authentication. Routine culture was performed using DMEM +GlutMAX™-I (ThermoFisher Scientific) supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin. MDA-MB-468 (human, female origin) were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin. U-2 OS (human, female origin) and MDA-MB-231 (human, female origin) were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin.
- A plasmid based CRISPR pooled lentiviral sgRNA library that can be used in a modular format was purchased from CELLECTA (CELLECTA: CRISPR Human Genome Knockout Library, Module 1 (200 µg plasmid), #sgRNA: 52,549, KOHGW-M1-P; Module 2 (200 µg plasmid), #sgRNA: 49,461, KOHGW-M2-P; and Module 3 (200 µg plasmid), #sgRNA: 48,578, KOHGW-M3-P). Each module covers approximately 6,300 genes and each gene is targeted by 8x sgRNAs (~50,000 sgRNAs per module), in total targeting >19,000 protein coding genes. Lentiviral production, functional titer estimation and pooled genome-wide CRISPR/Cas9 screening were performed according to user manual (CRISPR pooled lentiviral sgRNA libraries; www.cellecta.com) with minor modifications as outlined below.
- For lentivirus production, 25×106 Lenti-X 293T cells were seeded on 20 × 150 mm plates for each sgRNA library module and transfected the following day. For each module, a master stock of packaging plasmid (600 µg) and sgRNA plasmid (120 µg) were combined in 20 ml of 150 mM NaCl and mixed with 1440 µl of polyethylenimine (1 mg/ml) that had been diluted in 20 ml of 150 mM NaCl. After 20 minutes of
incubation 2 ml of the mix were added dropwise to each plate. 24 hours post-transfection, media was exchanged for DMEM medium supplemented with 10% FBS, DNase I (1 U/ml), 5 mM MgCl2, 20 mM HEPES pH7.4. Lentiviruses were harvested 48 hours post-transduction and filtered through a Nalgene 0.2 µm PES filter. Lentifuge (1 µl per ml) was added to the filtrate and incubated at 4 °C for 1 hour. The filtrate was then centrifuged for 2 hours at 12,000 rpm and the pellet resuspended in 1/100 of the original volume of D-PBS (Sigma), aliquoted and frozen at -80 °C. - Aliquots of viral supernatant were titered by adding incremental volumes (0.1 µl to 1 µl for concentrated virus or 10 µl to 100 µl for viral supernatant) in the presence of polybrene (5 µg/ml) to MCF10A EditR cells that had been seeded 24 hours in advance at 120,000 cells per well in a 6 well plate. Media was changed after 24 hours and at 48 hours after transduction, MCF10A EditR cells were harvested and fixed in 4% paraformaldehyde in PBS for 20 minutes at room temperature before being washed and resuspended in PBS. Number of RFP (Cellecta sgRNA library) expressing cells was determined on a
BD LSR FORTESSA 2 flow cytometer and analysis using FlowJo software. The number of transduction units per ml was determined using a titer chart (CELLECTA user manual: CRISPR pooled lentiviral sgRNA libraries; www.cellecta.com) with the following equation: TU per ml = (# of cells at Transduction) × [MOI / (ml of lentiviral stock used at transduction)]. - MCF10A EditR cells (120 × 106 per module) were transduced with individual modules (Modules I, II and III) in the presence of 5 µg/ml polybrene at a Multiplicity of Infection (Mol) of 0.4 achieving ~600-fold guide representation. After 24 hours, transduced cells were harvested, replated and selected with puromycin (1 µg/ml) for 6 days. Puromycin resistant cells were harvested and a sample of ~30 ×106 cells taken for analysis by next generation sequencing (NGS) at the start of the screen (day 0). The remaining puromycin resistant cells from each module were cultured independently from each other in untreated and XL413 treated (1.25 µM) experimental branches. Throughout the screen cell proliferation and viability were determined by cell counting on a LunaII automated cell counter (Logos Biosystems) using trypan blue exclusion. Cells were cultured, harvested, reseeded and retreated every 4 days in order to maintain ~600-fold guide representation. Finally at
day 20 for the untreated branch and atday 24 for the XL413-treated branche, cells were harvested and a sample of ~30 ×106 cells taken for analysis by NGS. - Cell pellets were resuspended in 6 ml of Buffer P1 (50 mM Tris-Cl, pH 8.0, 10 mM EDTA, Qiagen) and lysed by addition of SDS to final concentration of 1 % (v/v). Samples were sonicated at 10 % amplitude for 5 seconds (Branson, digital sonifier) before addition of proteinase K (40 µg/ml, Qiagen) followed by gentle mixing and incubation at 55°C overnight. Samples were cooled to 37°C, and following addition of RNaseA (100 µg/ml, Qiagen), incubated for 30min. Samples were chilled on ice before addition of 2 ml of ice cold 7.5 M ammonium acetate (Sigma) followed by two
repeat 20 second vortex-pulses and centrifugation at 4,000 g for 10 minutes. The supernatant was transferred to a 15 ml conical tube and genomic DNA was precipitated byaddition 6 ml of Isopropanol followed by mixing and centrifugation at 4,000 g for 40 minutes. Genomic DNA was washed with 6 ml of 70 % (v/v) Ethanol, centrifuged at 4,000 g for 10 minutes and the genomic DNA pellet air-dried before being resuspended in 220ul of DNase/RNase free water and quantitated on a Nanodrop. - Genomic DNA was subject to a first round PCR using multiple reactions, with a maximum of 50 µg of genomic DNA in each 100 µl PCR reaction, with 0.2 mM of each dNTP, 0.3 µM of each primer (Forward 5'-AGGGCCTATTTCCCATGATTCCT-3' and Reverse 5'-CGACAACAACGCAGAAATTTTGAAT-3'), and 1x Titanium Taq DNA polymerase in 1x Titanium Taq buffer. Genomic DNA was initially denatured at 94°C for 4 minutes, followed by 18 cycles of 94°C for 45 seconds, 63°C for 15 seconds, 72°C for 35 seconds, 1 cycle of 72°C for 4 minutes and hold at 4°C. All 1st round PCR reactions for a sample were pooled, mixed and a 5ml aliquot used directly for 2nd round PCR in a 100 µl final volume, as described before, except using 0.5 µM of each primer designed to add Illumina: flow-cell adaptors; barcodes and annealing sites for sequencing primers (NGS-Fwd1 5'-AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACA CGACGCTCTTCCGATCTTTGTGGAAAGGACGAAACACCG-3' and NGS-Rev2 5'-CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTG TGCTCTTCCGATCTGAGGTTCAGAGTTCTACAGTCC-3'). The 2nd round PCR was subject to PCR clean-up (Macherey-Nagel), to concentrate the DNA into a 50 µl volume, followed by gel purification (Macherey-Nagel) of a DNA product of ~293bp from a 2 % (w/v) agarose DNA gel. Gel purified libraries were sent to Novagene for ilummina NGS analysis using a NovaSeq 6000 PE150, paired-end reads and 30Gb of raw data per sample. The libraries were shipped to Novagene for ilummina NGS analysis using a NovaSeq 6000 PE150, with paired-end reads and 30 Gb raw data per sample.
- FASTQ files obtained by NGS were analysed using the MAGeCKFlute pipeline (Wang et al., 2019) as outlined below. MAGeCK was used to perform adaptor trimming, mapping of trimmed sgRNAs and associated sequencing reads to the CELLECTA sgRNA reference library, and a quality control assessment of the sequencing quality and screen data. Following quality control inspection, sgRNA read count data from the
day 0 vsday 20 untreated andday 0 vsday 24 XL413-treated (1.25 µM) conditions were modelled using a negative-binomial distribution by MAGeCK-MLE to calculate a beta score for each targeted gene. Negative control sgRNAs in the CELLECTA library were used by MAGeCK-MLE to normalize the read counts between samples. Beta score normalization was performed by MAGeCK-Flute, using the proposed list of core essential genes (Wang et al., 2019), to accommodate the effect of the CDC7 inhibitor on decreased cell proliferation throughout the screen. No copy number correction was applied as the MCF10A EditR cell line used in the screen are near diploid. The normalized control beta score was subtracted from normalized treatment beta score, for each gene target, and the genes were ranked and plotted based on this differential beta score. Gene hits were identified as outliers +/- 2x standard deviation of the mean differential beta score. For the hits identified, the normalised sgRNA count data was plotted and used to compare the relative abundance inday 20 untreated andday 24 XL413-treated (1.25 µM) samples today 0. - MCF10A cells were seeded at 4,000 cells per well of a 6 well plate and after 24 hours were treated with inhibitors before being incubated for 7 days at 37 °C with 5% CO2. Media was removed from the cells which were then washed with PBS and air dried before being fixed with 100 % methanol for 1 hour. Following removal of methanol the plates were air-dried, stained with crystal violet staining solution (0.2% crystal violet, 25% methanol) for 1 hour at room temperature, washed with ddH2O and air-dried. Plates were scanned using a HP Scanjet 4050, to generate images of crystal violet stained cells, and crystal violet staining intensity was acquired using the Odyssey infrared imaging system and Image Studio software (LI-COR Biosciences).
- To analyse the cell cycle and DNA synthesis, nascent DNA was labelled by incubating cells with 10 µM EdU for either 20 or 30 minutes prior to harvest, washing with PBS, and then by resuspension in PBS containing 70% EtOH for 1 hour at -20 °C. After a PBS wash, incorporated EdU was labelled by incubating cells in click reaction buffer (PBS containing 10 µM 6-Carboxyfluorescine-TEG-azide, 10 mM Sodium-L-ascorbate, 2 mM Copper-II-Sulphate) for 30 minutes protected from light. Cells were washed in PBS containing 1% BSA, 5
% Tween 20 and then DNA stained in PBS containing 1% BSA and 1 µg/ml DAPI. All washes involve: centrifugation at 400 x g for 5 minutes, removal of supernatant from pellet, resuspension in the appropriate buffer, recentrifugation and removal of supernatant from the pellet prior to the next step. - Fluorescence intensity data for 6-Carboxyfluorescine (488_530_30 nm) and DAPI (405_450_50 nm) were acquired for 10,000 single cells on a BD FACS Canto II and analysed using FlowJo software. To further analyse fluorescence intensity, proportional to EdU incorporation, for individual cells, gates were applied on DAPI-EdU biparametric dot plots to select for EdU positive S phase cells using FlowJo. According to the required analysis these gates included either all S phase cells, or only early or late S phase cells. Fluorescence intensity (488_530_30) values per cell were exported and plotted as scatter dot plots using GraphPad prism. For comparisons of mean fluorescence intensity of a cell population across three biological repeat experiments, all fluorescence intensity values were normalised to the maximum fluorescence intensity of the untreated control sample of each respective experiment to account for absolute differences in EdU staining. The mean fluorescence intensity values of these cell populations across three biological repeat experiments are expressed as a ratio relative to a control sample and plotted as a scatter dot plot using GraphPad prism.
- GraphPad PRISM 8.4.2 for windows was used to generate all graphs and perform statistical test. Information about the statistical tests and the number of repeat experiments analysed are provided in the figure legends.
-
- Adler, A.S., McCleland, M.L., Truong, T., Lau, S., Modrusan, Z., Soukup, T.M., Roose-Girma, M., Blackwood, E.M., and Firestein, R. (2012). CDK8 Maintains Tumor Dedifferentiation and Embryonic Stem Cell Pluripotency. Cancer Res. 72, 2129-2139. Akamatsu, M., Mikami, N., Ohkura, N., Kawakami, R., Kitagawa, Y., Sugimoto, A., Hirota, K., Nakamura, N., Ujihara, S., Kurosaki, T., et al. (2019). Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Tregcells by inhibition of CDK8/19. Sci. Immunol. 4.
- Alver, R.C., Chadha, G.S., Gillespie, P.J., and Blow, J.J. (2017). Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1. Cell Rep. 18, 2508.
- Arnett, A., Moo, K.G., Flynn, K.J., Sundberg, T.B., Johannessen, L., Shamji, A.F., Gray, N.S., Decker, T., Zheng, Y., Gersuk, V.H., et al. (2021). The Cyclin-Dependent Kinase 8 (CDK8) Inhibitor DCA Promotes a Tolerogenic Chemical Immunophenotype in CD4 + T Cells via a Novel CDK8-GATA3-FOXP3 Pathway . Mol. Cell. Biol. 41. Berge, S.M., Bighley, L.D., and Monkhouse, D.C. (1977). Pharmaceutical salts. J. Pharm. Sci. 66, 1-19.
- Boos, D., Yekezare, M., and Diffley, J.F.X. (2013). Identification of a heteromeric complex that promotes DNA replication origin firing in human cells. Science (80-. ). 340, 981-984.
- Chen, M., Li, J., Liang, J., Thompson, Z.S., Kathrein, K., Broude, E. V., and Roninson, I.B. (2019). Systemic Toxicity Reported for CDK8/19 Inhibitors CCT251921 and MSC2530818 Is Not Due to Target Inhibition. .
- Cheng, A.N., Lo, Y.K., Lin, Y.S., Tang, T.K., Hsu, C.H., Hsu, J.T.A., and Lee, A.Y.L. (2018). Identification of Novel Cdc7 Kinase Inhibitors as Anti-Cancer Agents that Target the Interaction with Dbf4 by the Fragment Complementation and Drug Repositioning Approach. EBioMedicine 36, 241.
- Clarke, P.A., Ortiz-Ruiz, M.-J., TePoele, R., Adeniji-Popoola, O., Box, G., Court, W., Czasch, S., El Bawab, S., Esdar, C., Ewan, K., et al. (2016). Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases. .
- Cowley, G.S., Weir, B.A., Vazquez, F., Tamayo, P., Scott, J.A., Rusin, S., East-Seletsky, A., Ali, L.D., Gerath, W.F.J., Pantel, S.E., et al. (2014). Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies. .
- Czodrowski, P., Mallinger, A., Wienke, D., Esdar, C., Pöschke, O., Busch, M., Rohdich, F., Eccles, S.A., Ortiz-Ruiz, M.J., Schneider, R., et al. (2016). Structure-Based Optimization of Potent, Selective, and Orally Bioavailable CDK8 Inhibitors Discovered by High-Throughput Screening. J. Med. Chem. 59, 9337-9349.
- Dannappel, M.V., Sooraj, D., Loh, J.J., Firestein, R., MV, D., D, S., JJ, L., and R, F. (2019). Molecular and in vivo Functions of the CDK8 and CDK19 Kinase Modules. Front. Cell Dev. Biol. 6, 171.
- Deady, L.W. (1977). Ring Nitrogen Oxidation of Amino Substituted Nitrogen Heterocycles with M-Chloroperbenzoic Acid. Synth. Commun. 7, 509-514.
- Ermoli, A., Bargiotti, A., Brasca, M.G., Ciavolella, A., Colombo, N., Fachin, G., Isacchi, A., Menichincheri, M., Molinari, A., Montagnoli, A., et al. (2009). Cell division cycle 7 kinase inhibitors: 1H-pyrrolo[2,3-b]pyridines, synthesis and structure-activity relationships. J. Med. Chem. 52, 4380-4390.
- Ettl, T., Schulz, D., and Bauer, R.J. (2022). The Renaissance of Cyclin Dependent Kinase Inhibitors. Cancers (Basel). 14.
- Fant, C.B., and Taatjes, D.J. (2019). Regulatory functions of the Mediator kinases CDK8 and CDK19. .
- Ferreira, P., Höfer, V., Kronshage, N., Marko, A., Reusswig, K.U., Tetik, B., Dieße1, C., Köhler, K., Tschernoster, N., Altmüller, J., et al. (2021). MTBP phosphorylation controls DNA replication origin firing. Sci. Rep. 11.
- Firestein, R., Bass, A.J., Kim, S.Y., Dunn, I.F., Silver, S.J., Guney, I., Freed, E., Ligon, A.H., Vena, N., Ogino, S., et al. (2008). CDK8 is a colorectal cancer oncogene that regulates β-catenin activity. Nature 455, 547-551.
- Firestein, R., Shima, K., Nosho, K., Irahara, N., Baba, Y., Bojarski, E., Giovannucci, E.L., Hahn, W.C., Fuchs, C.S., and Ogino, S. (2010). CDK8 expression in 470 colorectal cancers in relation to beta-catenin activation, other molecular alterations and patient survival. Int. J. Cancer 126, 2863-2873.
- Galbraith, M.D., Donner, A.J., and Espinosa, J.M. (2010). CDK8: A positive regulator of transcription. .
- Gilbert, D.M., Takebayashi, S.-I., Ryba, T., Lu, J., Pope, B.D., Wilson, K.A., and Hiratani, I. (2010). Space and Time in the Nucleus: Developmental Control of Replication Timing and Chromosome Architecture. Cold Spring Harb. Symp. Quant. Biol. 75, 143-153.
- Guo, Y., Wang, J., Benedict, B., Yang, C., van Gemert, F., Ma, X., Gao, D., Wang, H., Zhang, S., Lieftink, C., et al. (2021). Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer. Genome Med. 13.
- Harper, T.M., and Taatjes, D.J. (2018). The complex structure and function of Mediator. J. Biol. Chem. 293, 13778-13785.
- Hatcher, J.M., Wang, E.S., Johannessen, L., Kwiatkowski, N., Sim, T., and Gray, N.S. (2018). Development of Highly Potent and Selective Steroidal Inhibitors and Degraders of CDK8. ACS Med. Chem. Lett. 9, 540-545.
- Hatcher, J.M., Vatsan, P.S., Wang, E., Jiang, J., and Gray, N.S. (2021). Development of Highly Potent and Selective Pyrazolopyridine Inhibitor of CDK8/19. ACS Med. Chem. Lett. 12, 1689-1693.
- Hiraga, S.-I., Ly, T., Garzón, J., Hořejší, Z., Ohkubo, Y.-N., Endo, A., Obuse, C., Boulton, S.J., Lamond, A.I., and Donaldson, A.D. (2017). Human RIF1 and .
- Hofmann, M.H., Mani, R., Engelhardt, H., Impagnatiello, M.A., Carotta, S., Kerenyi, M., Lorenzo-Herrero, S., Böttcher, J., Scharn, D., Arnhof, H., et al. (2020). Selective and potent CDK8/19 inhibitors enhance NK-cell activity and promote tumor surveillance. Mol. Cancer Ther. 19, 1018-1030.
- Irie, T., Asami, T., Sawa, A., Uno, Y., Hanada, M., Taniyama, C., Funakoshi, Y., Masai, H., and Sawa, M. (2017). Discovery of novel furanone derivatives as potent Cdc7 kinase inhibitors. Eur. J. Med. Chem. 130, 406-418.
- Irie, T., Asami, T., Sawa, A., Uno, Y., Taniyama, C., Funakoshi, Y., Masai, H., and Sawa, M. (2021). Discovery of AS-0141, a Potent and Selective Inhibitor of CDC7 Kinase for the Treatment of Solid Cancers. J. Med. Chem. 64, 14153-14164.
- Iwai, K., Nambu, T., Dairiki, R., Ohori, M., Yu, J., Burke, K., Gotou, M., Yamamoto, Y., Ebara, S., Shibata, S., et al. (2019). Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor. Sci. Adv. 5.
- Iwai, K., Nambu, T., Kashima, Y., Yu, J., Eng, K., Miyamoto, K., Kakoi, K., Gotou, M., Takeuchi, T., Kogame, A., et al. (2021). A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery. Sci. Adv. 7, 197-218.
- Johannessen, L., Sundberg, T.B., O'Connell, D.J., Kolde, R., Berstler, J., Billings, K.J., Khor, B., Seashore-Ludlow, B., Fassl, A., Russell, C.N., et al. (2017). Small-molecule studies identify CDK8 as a regulator of IL-10 in myeloid cells. Nat. Chem. Biol. 13, 1102-1108.
- Köhler, K., Sanchez-Pulido, L., Höfer, V., Marko, A., Ponting, C.P., Snijders, A.P., Feederle, R., Schepers, A., and Boos, D. (2019). The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7.
- Koltun, E.S., Tsuhako, A.L., Brown, D.S., Aay, N., Arcalas, A., Chan, V., Du, H., Engst, S., Ferguson, K., Franzini, M., et al. (2012). Discovery of XL413, a potent and selective CDC7 inhibitor. Bioorg. Med. Chem. Lett. 22, 3727-3731.
- Kurasawa, O., Homma, M., Oguro, Y., Miyazaki, T., Mori, K., Uchiyama, N., Iwai, K., Ohashi, A., Hara, H., Yoshida, S., et al. (2017). 2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones bearing 3-methylpyrazole hinge binding moiety: Highly potent, selective, and time-dependent inhibitors of Cdc7 kinase. Bioorg. Med. Chem. 25, 3658-3670. Kurasawa, O., Miyazaki, T., Homma, M., Oguro, Y., Imada, T., Uchiyama, N., Iwai, K., Yamamoto, Y., Ohori, M., Hara, H., et al. (2020). Discovery of a Novel, Highly Potent, and Selective Thieno[3,2- d]pyrimidinone-Based Cdc7 Inhibitor with a Quinuclidine Moiety (TAK-931) as an Orally Active Investigational Antitumor Agent. J. Med. Chem. 63, 1084-1104.
- Labib, K. (2010). How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells? Genes Dev. 24, 1208-1219.
- Liang, J., Chen, M., Hughes, D., Chumanevich, A.A., Altilia, S., Kaza, V., Lim, C.U., Kiaris, H., Mythreye, K., Pena, M.M., et al. (2018). CDK8 Selectively Promotes the Growth of Colon Cancer Metastases in the Liver by Regulating Gene Expression of TIMP3 and Matrix Metalloproteinases. Cancer Res. 78, 6594-6606.
- Lloyd, R.L., Urban, V., Muñoz-Martínez, F., Ayestaran, I., Thomas, J.C., De Renty, C., O'Connor, M.J., Forment, J. V., Galanty, Y., and Jackson, S.P. (2021). Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress. Nucleic Acids Res. 49.
- Mallinger, A., Crumpler, S., Pichowicz, M., Waalboer, D., Stubbs, M., Adeniji-Popoola, O., Wood, B., Smith, E., Thai, C., Henley, A.T., et al. (2015). Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen. J. Med. Chem. 58, 1717-1735.
- Mallinger, A., Schiemann, K., Rink, C., Stieber, F., Calderini, M., Crumpler, S., Stubbs, M., Adeniji-Popoola, O., Poeschke, O., Busch, M., et al. (2016). Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19. J. Med. Chem. 59, 1078-1101. Masai, H., Taniyama, C., Ogino, K., Matsui, E., Kakusho, N., Matsumoto, S., Kim, J.-M., Ishii, A., Tanaka, T., Kobayashi, T., et al. (2006). Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin. J. Biol. Chem. 281, 39249-39261.
- McDermott, M.S.J., Chumanevich, A.A., Lim, C.U., Liang, J., Chen, M., Altilia, S., Oliver, D., Rae, J.M., Shtutman, M., Kiaris, H., et al. (2017). Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer. .
- McDonald, E.R., de Weck, A., Schlabach, M.R., Billy, E., Mavrakis, K.J., Hoffman, G.R., Belur, D., Castelletti, D., Frias, E., Gampa, K., et al. (2017). Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 170, 577-592.e10.
- McFarland, J.M., Ho, Z. V., Kugener, G., Dempster, J.M., Montgomery, P.G., Bryan, J.G., Krill-Burger, J.M., Green, T.M., Vazquez, F., Boehm, J.S., et al. (2018). Improved estimation of cancer dependencies from large-scale RNAi screens using model-based normalization and data integration. Nat. Commun. 9, 4610.
- Menichincheri, M., Bargiotti, A., Berthelsen, J., Bertrand, J.A., Bossi, R., Ciavolella, A., Cirla, A., Cristiani, C., Croci, V., D'Alessio, R., et al. (2009). First Cdc7 kinase inhibitors: pyrrolopyridinones as potent and orally active antitumor agents. 2. Lead discovery. J. Med. Chem. 52, 293-307.
- Menichincheri, M., Albanese, C., Alli, C., Ballinari, D., Bargiotti, A., Caldarelli, M., Ciavolella, A., Cirla, A., Colombo, M., Colotta, F., et al. (2010). Cdc7 kinase inhibitors: 5-heteroaryl-3-carboxamido-2-aryl pyrroles as potential antitumor agents. 1. Lead finding. J. Med. Chem. 53, 7296-7315.
- Menzl, I., Witalisz-Siepracka, A., and Sexl, V. (2019). CDK8-novel therapeutic opportunities. .
- Meyers, R.M., Bryan, J.G., McFarland, J.M., Weir, B.A., Sizemore, A.E., Xu, H., Dharia, N. V, Montgomery, P.G., Cowley, G.S., Pantel, S., et al. (2017). Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. Nat. Genet. 49, 1779-1784.
- Montagnoli, A., Tenca, P., Sola, F., Carpani, D., Brotherton, D., Albanese, C., and Santocanale, C. (2004). Cdc7 Inhibition Reveals a p53-Dependent Replication Checkpoint That Is Defective in Cancer Cells. Cancer Res. 64, 7110-7116. Montagnoli, A., Valsasina, B., Croci, V., Menichincheri, M., Rainoldi, S., Marchesi, V., Tibolla, M., Tenca, P., Brotherton, D., Albanese, C., et al. (2008). A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity. Nat. Chem. Biol. 4, 357-365.
- Montagnoli, A., Moll, J., and Colotta, F. (2010). Targeting cell division cycle 7 kinase: A new approach for cancer therapy. Clin. Cancer Res. 16, 4503-4508.
- Nakamura, A., Nakata, D., Kakoi, Y., Kunitomo, M., Murai, S., Ebara, S., Hata, A., and Hara, T. (2018). CDK8/19 inhibition induces premature G1/S transition and ATR-dependent cell death in prostate cancer cells. Oncotarget 9.
- Natoni, A., Murillo, L.S., Kliszczak, A.E., Catherwood, M.A., Montagnoli, A., Samali, A., O'Dwyer, M., and Santocanale, C. (2011). Mechanisms of action of a dual Cdc7/Cdk9 kinase inhibitor against quiescent and proliferating CLL cells. Mol. Cancer Ther. 10, 1624-1634.
- Natoni, A., Coyne, M.R.E., Jacobsen, A., Rainey, M.D., O'Brien, G., Healy, S., Montagnoli, A., Moll, J., O'Dwyer, M., and Santocanale, C. (2013). Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models. Cancers (Basel). 5, 901-918.
- Nemet, J., Jelicic, B., Rubelj, I., and Sopta, M. (2014). The two faces of Cdk8, a positive/negative regulator of transcription. Biochimie 97, 22-27.
- Pelish, H.E., Liau, B.B., Nitulescu, I.I., Tangpeerachaikul, A., Poss, Z.C., Da Silva, D.H., Caruso, B.T., Arefolov, A., Fadeyi, O., Christie, A.L., et al. (2015). Mediator kinase inhibition further activates super-enhancer-associated genes in AML. Nature 526, 273-276.
- Philip, S., Kumarasiri, M., Teo, T., Yu, M., and Wang, S. (2018). Cyclin-Dependent Kinase 8: A New Hope in Targeted Cancer Therapy? J. Med. Chem. 61, 5073-5092. Porter, D.C., Farmaki, E., Altilia, S., Schools, G.P., West, D.K., Chen, M., Chang, B.D., Puzyrev, A.T., Lim, C.U., Rokow-Kittell, R., et al. (2012). Cyclin-.
- Poss, Z.C., Ebmeier, C.C., Odell, A.T., Tangpeerachaikul, A., Lee, T., Pelish, H.E., Shair, M.D., Dowell, R.D., Old, W.M., and Taatjes, D.J. (2016). Identification of Mediator kinase substrates in human cells using cortistatin A and quantitative phosphoproteomics. Cell Rep. 15, 436.
- Putz, E.M., Gotthardt, D., Hoermann, G., Csiszar, A., Wirth, S., Berger, A., Straka, E., Rigler, D., Wallner, B., Jamieson, A.M., et al. (2013). CDK8-mediated STAT1-S727 phosphorylation restrains NK cell cytotoxicity and tumor surveillance. Cell Rep. 4, 437-444.
- Rainey, M.D., Quachthithu, H., Gaboriau, D., and Santocanale, C. (2017). DNA Replication Dynamics and Cellular Responses to ATP Competitive CDC7 Kinase Inhibitors. ACS Chem. Biol. 12, 1893-1902.
- Rainey, M.D., Bennett, D., O'Dea, R., Zanchetta, M.E., Voisin, M., Seoighe, C., and Santocanale, C. (2020). ATR Restrains DNA Synthesis and Mitotic Catastrophe in Response to CDC7 Inhibition. Cell Rep. 32.
- Remington, J.P. (Joseph P., and Osol, A. (1980). Remington's Pharmaceutical sciences, (ISBN: 9780912374024). 1928.
- Rzymski, T., Mikula, M., Zylkiewicz, E., Dreas, A., Wiklik, K., Golas, A., Wójcik, K., Masiejczyk, M., Wróbel, A., Dolata, I., et al. (2017). SEL120-34A is a novel CDK8 inhibitor active in AML cells with high levels of serine phosphorylation of STAT1 and STAT5 transactivation domains. .
- Sasi, N.K., Tiwari, K., Soon, F.F., Bonte, D., Wang, T., Melcher, K., Xu, H.E., and Weinreich, M. (2014). The potent Cdc7-Dbf4 (DDK) kinase inhibitor XL413 has limited activity in many cancer cell lines and discovery of potential new DDK inhibitor scaffolds. PLoS One 9.
- Sasi, N.K., Bhutkar, A., Lanning, N.J., MacKeigan, J.P., and Weinreich, M. (2017). DDK Promotes Tumor Chemoresistance and Survival via Multiple Pathways. Neoplasia 19, 439.
- Seo, J.O., Han, S.I., and Lim, S.C. (2010). Role of CDK8 and β-catenin in colorectal adenocarcinoma. Oncol. Rep. 24, 285-291.
- Sheu, Y.-J., and Stillman, B. (2006). Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. Mol. .
- Sheu, Y.-J., and Stillman, B. (2010). The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Nature 463, 113-117.
- Stahl, P.H., Wermuth, C.G., and International Union of Pure and Applied Chemistry. (2011). Handbook of pharmaceutical salts : properties, selection, and use. 446. Teesdale-Spittle, P. (1993). Advanced organic chemistry: Reactions, mechanisms and structure, 4th edn, Jerry March, Wiley, New York, 1992, 1512 pp. £24.95 (paperback), £45.50 (cloth). ISBN 0471581488. Appl. Organomet. Chem. 7, 293-293.
- Tsherniak, A., Vazquez, F., Montgomery, P.G., Weir, B.A., Kryukov, G., Cowley, G.S., Gill, S., Harrington, W.F., Pantel, S., Krill-Burger, J.M., et al. (2017). Defining a Cancer Dependency Map. Cell 170, 564-576.e16.
- Tsutsui, T., Umemura, H., Tanaka, A., Mizuki, F., Hirose, Y., and Ohkuma, Y. (2008). Human mediator kinase subunit CDK11 plays a negative role in viral activator VP16-dependent transcriptional regulation. Genes to Cells 13, 817-826.
- Vanotti, E., Amici, R., Bargiotti, A., Berthelsen, J., Bosotti, R., Ciavolella, A., Cirla, A., Cristiani, C., D'Alessio, R., Forte, B., et al. (2008). Cdc7 kinase inhibitors: pyrrolopyridinones as potential antitumor agents. 1. Synthesis and structure-activity relationships. J. Med. Chem. 51, 487-501.
- Wang, B., Wang, M., Zhang, W., Xiao, T., Chen, C.-H., Wu, A., Wu, F., Traugh, N., Wang, X., Li, Z., et al. (2019). Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat. Protoc. 14, 756-780.
- Wermuth, C.G., and Stahl, P.H. (2002). IUPAC Pharmaceutical Salts: Properties, Selection, and Use A Handbook. .
- Xi, M., Chen, T., Wu, C., Gao, X., Wu, Y., Luo, X., Du, K., Yu, L., Cai, T., Shen, R., et al. (2019). CDK8 as a therapeutic target for cancers and recent developments in discovery of CDK8 inhibitors. Eur. J. Med. Chem. 164, 77-91.
- Zhang, L., Cheng, C., Li, J., Wang, L., Chumanevich, A.A., Porter, D.C., Mindich, A., Gorbunova, S., Roninson, I.B., Chen, M., et al. (2022). A Selective and Orally Bioavailable Quinoline-6-Carbonitrile-Based Inhibitor of CDK8/19 Mediator Kinase with Tumor-Enriched Pharmacokinetics. J. Med. Chem. 65, 3420-3433.
- All references discussed herein are incorporated by reference.
- The mRNA sequences below represent the longest transcripts and encode the longest isoforms for: CDC7 (NM_003503.4), CDK8 (NM_001260.3) and CCNC (NM_005190.4), respectively. In each case the coding sequences is highlighted by an underline.
-
-
-
- Example target sequences for siRNA oligos specific for Cdc7 coding region include, but is not an exhaustive list: 5'-aagctcagcaggaaaggtgtt-3' (Cdc7-A); 5'-aaagaagttccagcacaagac-3' (Cdc7-B); and 5'-aagcagtcaaagactgtggat-3' (Cdc7-D) (Montagnoli et al., 2004).
- Example target sequences for siRNA oligos specific for CDK8 and CCNC coding region include, but is not an exhaustive list: 5'-GCAACCAUUCUUAGUAAGAUGCCAA-3' (CCNC #1), 5'-GGAAUUUCCUUAUAGGAUGAAUCAT-3' (CCNC #2), 5'-AGGCAUUAUACCAAACGUAUUGATA-3' (CDK8 #1), 5'-GUAAAGCAUUCCACUUGCUUCAGAA-3' (CDK8 #2) (Lloyd et al., 2021).
- Example target sequences for lentiviral shRNA constructs generated by the RNAi consortium (GPP Web Portal - Home (broadinstitute.org)). Some of these shRNA sequences have been used to target CDK8 and CCNC (Firestein et al., 2008) and are also commercially available through sigma-aldrich/Merck. This is not an exhaustive list.
Gene Symbol Taxon Gene ID Clone ID Target Seq CDC7 human 8317 TRCN0000350364 TTCAAGAAGTACGGGAATATA CDC7 human 8317 TRCN0000003172 CGCATTCATCAGTTTGGTATT CDC7 human 8317 TRCN0000320935 CGCATTCATCAGTTTGGTATT CDC7 human 8317 TRCN0000196970 GCTGACAGATTTCCCATTTAG CDC7 human 8317 TRCN0000320938 AGAGACCAGAGCAGGATTAAT CDC7 human 8317 TRCN0000382344 GCTGTACCACAGCTTAGTAAT CDC7 human 8317 TRCN0000195201 CCAATCAAACTACAGCAATTG CDC7 human 8317 TRCN0000196542 GAAGCTTTGTTGCATCCATTT CDC7 human 8317 TRCN0000320936 GAAGCTTTGTTGCATCCATTT CDC7 human 8317 TRCN0000003171 CCTAATCTGTTTGGTAAGTAT CDC7 human 8317 TRCN0000003169 CGTGATGTTAAGCCCAGCAAT CDC7 human 8317 TRCN0000003168 GCCACAGCACAGTTACAAGTA CDC7 human 8317 TRCN0000320863 GCCACAGCACAGTTACAAGTA CDC7 human 8317 TRCN0000003170 CCAGGACAATACTCAGGGAAT CDC7 human 8317 TRCN0000196543 GAATACAGAATTGACGGTATT CDK8 human 1024 TRCN0000382350 ATGGTGAAGTCACTATTATAT CDK8 human 1024 TRCN0000322391 CATGGGCTTTGCCCGATTATT CDK8 human 1024 TRCN0000322410 CTAACGTCAGAACCAATATTT CDK8 human 1024 TRCN0000350308 CTAACGTCAGAACCAATATTT CDK8 human 1024 TRCN0000196569 GAATGGTGAAGTCACTATTAT CDK8 human 1024 TRCN0000350344 GCTTACCATGGACCCAATAAA CDK8 human 1024 TRCN0000195594 CACTACCTCAGGTGGACTTAT CDK8 human 1024 TRCN0000322329 TTTGGGCTATAGGGTGTATAT CDK8 human 1024 TRCN0000199980 GTCCATGCACTGTTGCGAATG CDK8 human 1024 TRCN0000196702 GCTATTGATATTTGGGCTATA CDK8 human 1024 TRCN0000000493 CACTTCCTACATCAGACGTTT CDK8 human 1024 TRCN0000362345 TATATTTGCAGAACTACTAAC CDK8 human 1024 TRCN0000195463 CGAGAGCTTAAGCATCCAAAC CDK8 human 1024 TRCN0000000490 GCAGGGCAATAACCACACTAA CDK8 human 1024 TRCN0000320651 GCAGGGCAATAACCACACTAA CDK8 human 1024 TRCN0000194708 CATGAGAATGTACTGTACAAC CDK8 human 1024 TRCN0000000491 CCTCTGGCATATAATCAAGTT CDK8 human 1024 TRCN0000023264 GCCTTATCAAGTATATGGAAA CDK8 human 1024 TRCN0000023108 GCAGATAAAGATTGGGAAGAT CDK8 human 1024 TRCN0000023106 CCCGATTATTTAATTCACCTT CDK8 human 1024 TRCN0000000492 CAAGGCATTATACCAAAGCTA CDK8 human 1024 TRCN0000000489 ATGTCCAGTAGCCAAGTTCCA CDK8 human 1024 TRCN0000320652 ATGTCCAGTAGCCAAGTTCCA CDK8 human 1024 TRCN0000199779 GAACCTGGTATGGGCCATGAG CCNC human 892 TRCN0000304592 TCTAGCTACAGCCAATCTTAA CCNC human 892 TRCN0000077831 GCATTAGGTGAACATCTTAAA CCNC human 892 TRCN0000020189 GCATCCAAAGTAGAGGAATTT CCNC human 892 TRCN0000319164 GCATCCAAAGTAGAGGAATTT CCNC human 892 TRCN0000020193 CCCACTATTTGCAATGGATTT CCNC human 892 TRCN0000077830 CATGGAGGATAGTGAATGATA CCNC human 892 TRCN0000302106 CATGGAGGATAGTGAATGATA CCNC human 892 TRCN0000020191 GCATGGAGGATAGTGAATGAT CCNC human 892 TRCN0000319165 GCATGGAGGATAGTGAATGAT CCNC human 892 TRCN0000020190 CCATTCTTAGTAAGATGCCAA CCNC human 892 TRCN0000319166 CCATTCTTAGTAAGATGCCAA CCNC human 892 TRCN0000020192 CTACGGTATATTTCAAGAGAT CCNC human 892 TRCN0000319188 CTACGGTATATTTCAAGAGAT
Claims (14)
- A Cell Division Cycle 7 kinase inhibitor (CDC7i) used in a combination therapy with:i) a Cyclin Dependent Kinase 8 inhibitor (CDK8i), and/orii) a Cyclin C inhibitor (CCNCi);
for use in the treatment or prevention of cancer in a subject. - The CDC7i for the use of claim 1, wherein the CDC7i comprises or consists of any one of the group of CDC7 inhibitors selected from PHA-767491 (Montagnoli et al., 2008), XL413, AS-0141, Dequalinium Chloride, LY3143921, TQB3824, NMS-1116354, and TAK-931; or variants or derivatives thereof; and/or combinations thereof; or the CDC7i is an inhibitory RNA molecule capable of inhibiting the expression of CDC7.
- The CDC7i for the use of claim 1 or claim 2, wherein the CDK8i comprises or consists of any one of the group of CDK8 inhibitors selected from BI1347, RVU120, Senexin A, Senexin B, Senexin C, MSC2530818, AS2863619, 16-didehydro-cortistatin A, BRD6989, CCT-251545, CCT-251921, TSN084, JH-XVI-178, and JH-XI-10-02; or variants or derivatives thereof; and/or combinations thereof; or the CDK8i is an inhibitory RNA molecule capable of inhibiting the expression of CDK8.
- The CDC7i for the use according to any preceding claim, wherein the CCNCi is an inhibitory RNA molecule capable of inhibiting the expression of CCNC.
- A composition comprising a CDC7 inhibitor and a CDK8 and/or CCNC inhibitor.
- A kit, wherein the kit comprises:i) a CDC7 inhibitor; andii) a CDK8 and/or CCNC inhibitor.
- A composition according to claim 5 or kit according to claim 6 for use as a medicament.
- The composition or kit for the use according to claim 7, wherein the use is for treatment or prevention of cancer in a subject.
- A Cell Division Cycle 7 kinase inhibitor (CDC7i) for use in a method of treatment or prevention of cancer in a subject, wherein the cancer is determined or known to be deficient in functional CDK8 and/or CNCC expression.
- The CDC7i for the use according to claim 9, wherein the cancer is selected from Diffuse Large B-Cell Lymphoma; Sarcoma; Lung Adenocarcinoma; Prostate Adenocarcinoma; Uveal Melanoma; Liver Hepatocellular Carcinoma; Stomach Adenocarcinoma; and Bladder Urothelial Carcinoma.
- Use of CDK8 and/or CCNC as a biomarker for predicting the efficacy of CDC7i therapy for cancer of a subject, wherein the reduction or absence of expression of functional CDK8 and/or CCNC in a subject is indicative of an increased efficacy of the CDC7i therapy for cancer.
- A method of predicting the efficacy of a CDC7i therapy for cancer in a subject, the method comprising determining the levels of expression of functional CDK8 and/or CCNC in the subject, or obtaining results of such a determination,
wherein a decreased level of expression of CDK8 and/or CCNC in the subject is indicative of a more effective CDC7i therapy for the cancer in the subject. - A method of selecting a subject for treatment for cancer with a CDC7i, the method comprising determining the levels of expression of functional CDK8 and/or CCNC in the subject, or obtaining results of such a determination,
wherein the subject is selected for CDC7i treatment if they have a decreased level of expression of CDK8 and/or CCNC. - A method of treatment of a subject for cancer, the method comprising:- determining the levels of expression of functional CDK8 and/or CCNC in the subject, or- having obtained results of the levels of expression of functional CDK8 and/or CCNC in the subject; and- administering CDC7i treatment if the subject has a decreased level of expression of CDK8 and/or CCNC.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP22172868.6A EP4275686A1 (en) | 2022-05-11 | 2022-05-11 | Combination therapy for cancer comprising a cdc7 inhibitor and a cdk8 inihibitor or a ccnc inhibitor |
PCT/EP2023/062643 WO2023217980A1 (en) | 2022-05-11 | 2023-05-11 | Combination therapy for cancer comprising a cdc7 inhibitor and a cdk8 in i hibitor or a ccnc inhibitor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP22172868.6A EP4275686A1 (en) | 2022-05-11 | 2022-05-11 | Combination therapy for cancer comprising a cdc7 inhibitor and a cdk8 inihibitor or a ccnc inhibitor |
Publications (1)
Publication Number | Publication Date |
---|---|
EP4275686A1 true EP4275686A1 (en) | 2023-11-15 |
Family
ID=82115720
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP22172868.6A Pending EP4275686A1 (en) | 2022-05-11 | 2022-05-11 | Combination therapy for cancer comprising a cdc7 inhibitor and a cdk8 inihibitor or a ccnc inhibitor |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP4275686A1 (en) |
WO (1) | WO2023217980A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019165473A1 (en) * | 2018-02-26 | 2019-08-29 | Sierra Oncology, Inc | Methods of treatment of cancer comprising cdc7 inhibitors |
-
2022
- 2022-05-11 EP EP22172868.6A patent/EP4275686A1/en active Pending
-
2023
- 2023-05-11 WO PCT/EP2023/062643 patent/WO2023217980A1/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019165473A1 (en) * | 2018-02-26 | 2019-08-29 | Sierra Oncology, Inc | Methods of treatment of cancer comprising cdc7 inhibitors |
Non-Patent Citations (81)
Title |
---|
"NCBI", Database accession no. NM 005190.4 |
ADLER, A.SMCCLELAND, M.L.TRUONG, T.LAU, S.MODRUSAN, Z.SOUKUP, T.M.ROOSE-GIRMA, M.BLACKWOOD, E.M.FIRESTEIN, R: "CDK8 Maintains Tumor Dedifferentiation and Embryonic Stem Cell Pluripotency", CANCER RES., vol. 72, 2012, pages 2129 - 2139, XP055082310, DOI: 10.1158/0008-5472.CAN-11-3886 |
AKAMATSU, M.MIKAMI, N.OHKURA, N.KAWAKAMI, R.KITAGAWA, Y.SUGIMOTO, A.HIROTA, K.NAKAMURA, N.UJIHARA, SKUROSAKI, T ET AL.: "Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Tregcells by inhibition of CDK8/19", SCI. IMMUNOL., vol. 4, 2019 |
ALVER, R.C.CHADHA, G.S.GILLESPIE, P.J.BLOW, J.J: "Reversal of DDK-Mediated MCM Phosphorylation by Rifl-PPl Regulates Replication Initiation and Replisome Stability Independently of ATR/Chkl", CELL REP., vol. 18, 2017, pages 2508 |
ARNETT, A.MOO, K.G.FLYNN, K.J.SUNDBERG, T.B.JOHANNESSEN, L.SHAMJI, A.F.GRAY, N.S.DECKER, T., ZHENG, Y.GERSUK, V.H ET AL.: "The Cyclin-Dependent Kinase 8 (CDK8) Inhibitor DCA Promotes a Tolerogenic Chemical Immunophenotype in CD4 + T Cells via a Novel CDK8-GATA3-FOXP3 Pathway", MOL. CELL. BIOL., vol. 41, 2021 |
BERGE, S.M.BIGHLEY, L.DMONKHOUSE, D.C: "Pharmaceutical salts", J. PHARM. SCI., vol. 66, 1977, pages 1 - 19, XP002675560, DOI: 10.1002/jps.2600660104 |
BOOS, D.YEKEZARE, M.DIFFLEY, J.F.X.: "Identification of a heteromeric complex that promotes DNA replication origin firing in human cells", SCIENCE, vol. 340, 2013, pages 981 - 984 |
CHEN, M.LI, J.LIANG, J.THOMPSON, Z.S.KATHREIN, K.BROUDE, E. VRONINSON, I.B.: "Systemic Toxicity Reported for CDK8/19 Inhibitors CCT251921 and MSC2530818 Is Not Due to Target Inhibition", CELLS, vol. 8, 2019 |
CHENG, A.NLO, Y.K.LIN, Y.S.TANG, T.K.HSU, C.HHSU, J.T.A.LEE, A.Y.L.: "Identification of Novel Cdc7 Kinase Inhibitors as Anti-Cancer Agents that Target the Interaction with Dbf4 by the Fragment Complementation and Drug Repositioning Approach", EBIOMEDICINE, vol. 36, 2018, pages 241 |
CLARKE, P.A.ORTIZ-RUIZ, M.-J.TEPOELE, R.ADENIJI-POPOOLA, O.BOX, G.COURT, W.CZASCH, S.EL BAWAB, SESDAR, C.EWAN, K. ET AL.: "Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases", ELIFE, vol. 5, 2016 |
COWLEY, G.S.WEIR, B.A.VAZQUEZ, F.TAMAYO, P.SCOTT, J.A.RUSIN, S.EAST-SELETSKY, A.ALI, L.D.GERATH, W.F.J.PANTEL, S.E. ET AL.: "Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies", SCI. DATA, vol. 1, 2014, pages 1 - 12 |
CZODROWSKI, P.MALLINGER, A.WIENKE, D.ESDAR, C.POSCHKE, O.BUSCH, M.ROHDICH, FECCLES, S.A.ORTIZ-RUIZ, M.J.SCHNEIDER, R. ET AL.: "Structure-Based Optimization of Potent, Selective, and Orally Bioavailable CDK8 Inhibitors Discovered by High-Throughput Screening", J. MED. CHEM., vol. 59, 2016, pages 9337 - 9349, XP055744511, DOI: 10.1021/acs.jmedchem.6b00597 |
DANNAPPEL, M.V.SOORAJ, D.LOH, J.J.FIRESTEIN, R.MV, D.D, S.JJ, L.: "Molecular and in vivo Functions of the CDK8 and CDK19 Kinase Modules", FRONT. CELL DEV. BIOL., vol. 6, 2019, pages 171 |
DEADY, L.W: "Ring Nitrogen Oxidation of Amino Substituted Nitrogen Heterocycles with M-Chloroperbenzoic Acid", SYNTH. COMMUN., vol. 7, 1977, pages 509 - 514, XP055614111, DOI: 10.1080/00397917709409270 |
ERMOLI, ABARGIOTTI, A.BRASCA, M.G.CIAVOLELLA, A.COLOMBO, N.FACHIN, G.ISACCHI, A.MENICHINCHERI, MMOLINARI, A.MONTAGNOLI, A. ET AL.: "Cell division cycle 7 kinase inhibitors: 1H -pyrrolo [2,3 -b ]pyridines, synthesis and structure-activity relationships", J. MED. CHEM., vol. 52, 2009, pages 4380 - 4390, XP002637958, DOI: 10.1021/jm900248g |
ETTL, T.SCHULZ, D.BAUER, R.J: "The Renaissance of Cyclin Dependent Kinase Inhibitors", CANCERS (BASEL, vol. 14, 2022 |
FANT, C.B.TAATJES, D.J.: "Regulatory functions of the Mediator kinases CDK8 and CDK19", TRANSCRIPTION, vol. 10, 2019, pages 76 - 90 |
FERREIRA, PHOFER, V.KRONSHAGE, NMARKO, A.REUSSWIG, K.U.TETIK, BDIEBEL, CKOHLER, K.TSCHERNOSTER, N.ALTMIILLER, J. ET AL.: "MTBP phosphorylation controls DNA replication origin firing", SCI. REP., vol. 11, 2021 |
FIRESTEIN, R.BASS, A.J.KIM, S.Y.DUNN, I.F.SILVER, S.J.GUNEY, I.FREED, E.LIGON, A.HVENA, N.OGINO, S. ET AL.: "CDK8 is a colorectal cancer oncogene that regulates (3-catenin activity", NATURE, vol. 455, 2008, pages 547 - 551, XP055124701, DOI: 10.1038/nature07179 |
FIRESTEIN, R.SHIMA, K.NOSHO, K.IRAHARA, N.BABA, YBOJARSKI, E.GIOVANNUCCI, E.L.HAHN, W.C.FUCHS, C.S.OGINO, S.: "CDK8 expression in 470 colorectal cancers in relation to beta-catenin activation, other molecular alterations and patient survival", INT. J. CANCER, vol. 126, 2010, pages 2863 - 2873 |
GALBRAITH, M.D.DONNER, A.J.ESPINOSA, J.M.: "CDK8: A positive regulator of transcription", TRANSCRIPTION, vol. 1, 2010, pages 4 - 12 |
GILBERT, D.M.TAKEBAYASHI, S.-I.RYBA, T.LU, J.POPE, B.D.WILSON, K.A.HIRATANI, I: "Space and Time in the Nucleus: Developmental Control of Replication Timing and Chromosome Architecture", COLD SPRING HARB. SYMP. QUANT. BIOL., vol. 75, 2010, pages 143 - 153 |
GUO, Y.WANG, J.BENEDICT, B.YANG, C.VAN GEMERT, F.MA, X.GAO, D.WANG, H.ZHANG, SLIEFTINK, C. ET AL.: "Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer", GENOME MED, vol. 13, 2021, XP055921479, DOI: 10.1186/s13073-021-00981-0 |
HARPER, T.M.TAATJES, D.J.: "The complex structure and function of Mediator", J. BIOL. CHEM., vol. 293, 2018, pages 13778 - 13785 |
HATCHER, J.M.VATSAN, P.S.WANG, E.JIANG, J.GRAY, N.S.: "Development of Highly Potent and Selective Pyrazolopyridine Inhibitor of CDK8/19", ACS MED. CHEM. LETT., vol. 12, 2021, pages 1689 - 1693 |
HATCHER, J.M.WANG, E.S.JOHANNESSEN, LKWIATKOWSKI, N.SIM, T.GRAY, N.S.: "Development of Highly Potent and Selective Steroidal Inhibitors and Degraders of CDK8", ACS MED. CHEM. LETT, vol. 9, 2018, pages 540 - 545, XP055631975, DOI: 10.1021/acsmedchemlett.8b00011 |
HIRAGA, S.-I.LY, T.GARZON, J.HOREJSI, Z.OHKUBO, Y.-N.ENDO, A.OBUSE, C.BOULTON, S.J.LAMOND, A.IDONALDSON, A.D.: "Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation", EMBO REP, vol. 18, 2017, pages 403 - 419 |
HOFMANN MARCO H. ET AL: "Selective and Potent CDK8/19 Inhibitors Enhance NK-Cell Activity and Promote Tumor Surveillance", MOLECULAR CANCER THERAPEUTICS, vol. 19, no. 4, 1 April 2020 (2020-04-01), US, pages 1018 - 1030, XP055976400, ISSN: 1535-7163, Retrieved from the Internet <URL:https://aacrjournals.org/mct/article-pdf/19/4/1018/1849306/1018.pdf> DOI: 10.1158/1535-7163.MCT-19-0789 * |
HOFMANN, M.HMANI, R.ENGELHARDT, H.IMPAGNATIELLO, M.A.CAROTTA, S.KERENYI, M.LORENZO-HERRERO, S.BOTTCHER, J.SCHARN, DARNHOF, H. ET A: "Selective and potent CDK8/19 inhibitors enhance NK-cell activity and promote tumor surveillance", MOL. CANCER THER., vol. 19, 2020, pages 1018 - 1030 |
IRIE, T.ASAMI, T.SAWA, A.UNO, Y.TANIYAMA, CFUNAKOSHI, Y.MASAI, HSAWA, M.: "Discovery of AS-0141, a Potent and Selective Inhibitor of CDC7 Kinase for the Treatment of Solid Cancers", J. MED. CHEM., vol. 64, 2021, pages 14153 - 14164, XP055920957, DOI: 10.1021/acs.jmedchem.1c01319 |
IRIE, TASAMI, T.SAWA, A.UNO, Y.HANADA, M.TANIYAMA, C.FUNAKOSHI, Y.MASAI, H.SAWA, M.: "Discovery of novel furanone derivatives as potent Cdc7 kinase inhibitors", EUR. J. MED. CHEM., vol. 130, 2017, pages 406 - 418, XP029945186, DOI: 10.1016/j.ejmech.2017.02.030 |
IWAI, K.NAMBU, T.DAIRIKI, R.OHORI, MYU, J.BURKE, K.GOTOU, M.YAMAMOTO, Y.EBARA, S.SHIBATA, S. ET AL.: "Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor", SCI. ADV., vol. 5, 2019, XP055742770, DOI: 10.1126/sciadv.aav3660 |
IWAI, K.NAMBU, T.KASHIMA, YYU, J.ENG, K.MIYAMOTO, K.KAKOI, K.GOTOU, M.TAKEUCHI, T.KOGAME, A. ET AL.: "A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery", SCI. ADV., vol. 7, 2021, pages 197 - 218 |
JOHANNESSEN, LSUNDBERG, T.B.O'CONNELL, D.J.KOLDE, R.BERSTLER, J.BILLINGS, K.J.KHOR, B.SEASHORE-LUDLOW, B.FASSL, A.RUSSELL, C.N ET : "Small-molecule studies identify CDK8 as a regulator of IL-10 in myeloid cells", NAT. CHEM. BIOL., vol. 13, 2017, pages 1102 - 1108 |
KOHLER, K.SANCHEZ-PULIDO, L.HOFER, V.MARKO, A.PONTING, C.P.SNIJDERS, A.PFEEDERLE, R.SCHEPERS, A.BOOS, D, THE CDK8/19-CYCLIN C TRANSCRIPTION REGULATOR FUNCTIONS IN GENOME REPLICATION THROUGH METAZOAN SLD7, 2019 |
KOLTUN, E.S.TSUHAKO, A.L.BROWN, D.S.AAY, N.ARCALAS, A.CHAN, V.DU, H.ENGST, S.FERGUSON, K.FRANZINI, M. ET AL.: "Discovery of XL413, a potent and selective CDC7 inhibitor", BIOORG. MED. CHEM. LETT., vol. 22, 2012, pages 3727 - 3731, XP028423352, DOI: 10.1016/j.bmcl.2012.04.024 |
KURASAWA, O.HOMMA, M.OGURO, Y.MIYAZAKI, T.MORI, K.UCHIYAMA, N.IWAI, K.OHASHI, A.HARA, H.YOSHIDA, S. ET AL.: "2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones bearing 3-methylpyrazole hinge binding moiety: Highly potent, selective, and time-dependent inhibitors of Cdc7 kinase", BIOORG. MED. CHEM., vol. 25, 2017, pages 3658 - 3670, XP085062441, DOI: 10.1016/j.bmc.2017.04.044 |
KURASAWA, O.MIYAZAKI, T.HOMMA, M.OGURO, YIMADA, T.UCHIYAMA, N.IWAI, KYAMAMOTO, Y.OHORI, M.HARA, H. ET AL.: "Discovery of a Novel, Highly Potent, and Selective Thieno[3,2- d]pyrimidinone-Based Cdc7 Inhibitor with a Quinuclidine Moiety (TAK-931) as an Orally Active Investigational Antitumor Agent", J. MED. CHEM., vol. 63, 2020, pages 1084 - 1104, XP055742269, DOI: 10.1021/acs.jmedchem.9b01427 |
LABIB, K.: "How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?", GENES DEV., vol. 24, 2010, pages 1208 - 1219 |
LIANG, J., CHEN, M., HUGHES, D., CHUMANEVICH, A.A., ALTILIA, S., KAZA, V., LIM, C.U., KIARIS, H., MYTHREYE, K., PENA, M.M.: "CDK8 Selectively Promotes the Growth of Colon Cancer Metastases in the Liver by Regulating Gene Expression of TIMP3 and Matrix Metalloproteinases", CANCER RES., vol. 78, 2018, pages 6594 - 6606 |
LLOYD, R.L.URBAN, V.MUNOZ-MARTINEZ, F.AYESTARAN, I.THOMAS, J.CDE RENTY, C.O'CONNOR, M.J.FORMENT, J. V.GALANTY, Y.JACKSON, S.P.: "Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress", NUCLEIC ACIDS RES., vol. 49, 2021 |
MALLINGER, A.CRUMPLER, S.PICHOWICZ, M.WAALBOER, D.STUBBS, M.ADENIJI-POPOOLA, O.WOOD, B.SMITH, E.THAI, C.HENLEY, A.T. ET AL.: "Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen", J. MED. CHEM., vol. 58, 2015, pages 1717 - 1735, XP055188627, DOI: 10.1021/jm501436m |
MALLINGER, A.SCHIEMANN, K.RINK, CSTIEBER, F.CALDERINI, M.CRUMPLER, S.STUBBS, M.ADENIJI-POPOOLA, OPOESCHKE, OBUSCH, M. ET AL.: "Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19", J. MED. CHEM., vol. 59, 2016, pages 1078 - 1101, XP055336343, DOI: 10.1021/acs.jmedchem.5b01685 |
MASAI, H.TANIYAMA, C.OGINO, K.MATSUI, E.KAKUSHO, N.MATSUMOTO, S.KIM, J.-M.ISHII, A.TANAKA, T.KOBAYASHI, T. ET AL.: "Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin", J. BIOL. CHEM., vol. 281, 2006, pages 39249 - 39261 |
MCDERMOTT, M.S.J.CHUMANEVICH, A.A.LIM, C.ULIANG, J.CHEN, M.ALTILIA, S.OLIVER, DRAE, J.M.SHTUTMAN, M.KIARIS, H. ET AL.: "Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer", ONCOTARGET, vol. 8, 2017, pages 12558 - 12575, XP055445552, DOI: 10.18632/oncotarget.14894 |
MCDONALD, E.R.DE WECK, A.SCHLABACH, M.R.BILLY, E.MAVRAKIS, K.JHOFFMAN, G.R.BELUR, D.CASTELLETTI, D.FRIAS, E.GAMPA, K. ET AL.: "Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening", CELL, vol. 170, 2017, pages 577 - 592 |
MCFARLAND, J.M.HO, Z. V.KUGENER, G.DEMPSTER, J.M.MONTGOMERY, P.G.BRYAN, J.G.KRILL-BURGER, J.M.GREEN, T.M.VAZQUEZ, F.BOEHM, J.S. ET: "Improved estimation of cancer dependencies from large-scale RNAi screens using model-based normalization and data integration", NAT. COMMUN., vol. 9, 2018, pages 4610 |
MENICHINCHERI, M.ALBANESE, C.ALLI, C.BALLINARI, D.BARGIOTTI, ACALDARELLI, M.CIAVOLELLA, A.CIRLA, A.COLOMBO, M.COLOTTA, F. ET AL.: "Cdc7 kinase inhibitors: 5-heteroaryl-3-carboxamido-2-aryl pyrroles as potential antitumor agents. 1. Lead finding", J. MED. CHEM., vol. 53, 2010, pages 7296 - 7315, XP002619675, DOI: 10.1021/JM100504D |
MENICHINCHERI, M.BARGIOTTI, A.BERTHELSEN, J.BERTRAND, J.A.BOSSI, R.CIAVOLELLA, A.CIRLA, A.CRISTIANI, C.CROCI, V.D'ALESSIO, R ET AL: "First Cdc7 kinase inhibitors: pyrrolopyridinones as potent and orally active antitumor agents. 2. Lead discovery", J. MED. CHEM., vol. 52, 2009, pages 293 - 307 |
MENZL, I.WITALISZ-SIEPRACKA, A.SEXL, V.: "CDK8-novel therapeutic opportunities", PHARMACEUTICALS, vol. 12, 2019 |
MEYERS, R.M.BRYAN, J.G.MCFARLAND, J.M.WEIR, B.A.SIZEMORE, A.E.XU, H.DHARIA, N. VMONTGOMERY, P.G.COWLEY, G.S.PANTEL, S ET AL.: "Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells", NAT. GENET., vol. 49, 2017, pages 1779 - 1784 |
MONTAGNOLI, A.MOLL, J.COLOTTA, F: "Targeting cell division cycle 7 kinase: A new approach for cancer therapy", CLIN. CANCER RES., vol. 16, 2010, pages 4503 - 4508, XP055425979, DOI: 10.1158/1078-0432.CCR-10-0185 |
MONTAGNOLI, A.TENCA, PSOLA, F.CARPANI, D.BROTHERTON, D.ALBANESE, CSANTOCANALE, C.: "Cdc7 Inhibition Reveals a p53-Dependent Replication Checkpoint That Is Defective in Cancer Cells", CANCER RES., vol. 64, 2004, pages 7110 - 7116 |
MONTAGNOLI, A.VALSASINA, B.CROCI, V.MENICHINCHERI, M.RAINOLDI, S.MARCHESI, V.TIBOLLA, M.TENCA, P.BROTHERTON, D.ALBANESE, C. ET AL.: "A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity", NAT. CHEM. BIOL., vol. 4, 2008, pages 357 - 365 |
NAKAMURA, A.NAKATA, D.KAKOI, Y.KUNITOMO, MMURAI, S.EBARA, SHATA, A.HARA, T.: "CDK8/19 inhibition induces premature Gl/S transition and ATR-dependent cell death in prostate cancer cells", ONCOTARGET, vol. 9, 2018 |
NATONI, A.COYNE, M.R.E.JACOBSEN, A.RAINEY, M.D.O'BRIEN, G.HEALY, SMONTAGNOLI, A.MOLL, J.O'DWYER, M.SANTOCANALE, C.: "Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models", CANCERS (BASEL, vol. 5, 2013, pages 901 - 918 |
NATONI, A.MURILLO, L.S.KLISZCZAK, A.E.CATHERWOOD, M.A.MONTAGNOLI, A.SAMALI, A.O'DWYER, M.SANTOCANALE, C.: "Mechanisms of action of a dual Cdc7/Cdk9 kinase inhibitor against quiescent and proliferating CLL cells", MOL. CANCER THER., vol. 10, 2011, pages 1624 - 1634, XP055857737, DOI: 10.1158/1535-7163.MCT-10-1119 |
NEMET, J.JELICIC, B.RUBELJ, I.SOPTA, M: "The two faces of Cdk8, a positive/negative regulator of transcription", BIOCHIMIE, vol. 97, 2014, pages 22 - 27, XP028671647, DOI: 10.1016/j.biochi.2013.10.004 |
PELISH, H.E.LIAU, B.B.NITULESCU, I.ITANGPEERACHAIKUL, A.POSS, Z.C.DA SILVA, D.H.CARUSO, B.T.AREFOLOV, A.FADEYI, O.CHRISTIE, A.L. E: "Mediator kinase inhibition further activates super-enhancer-associated genes in AML", NATURE, vol. 526, 2015, pages 273 - 276, XP055328581, DOI: 10.1038/nature14904 |
PHILIP, S.KUMARASIRI, M.TEO, T.YU, M.WANG, S.: "Cyclin-Dependent Kinase 8: A New Hope in Targeted Cancer Therapy?", J. MED. CHEM., vol. 61, 2018, pages 5073 - 5092 |
PORTER, D.C.FARMAKI, E.ALTILIA, S.SCHOOLS, G.P.WEST, D.K.CHEN, MCHANG, B.D.PUZYREV, A.T.LIM, C.UROKOW-KITTELL, R. ET AL.: "Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities", PROC. NATL. ACAD. SCI. U. S. A., vol. 109, 2012, pages 13799 - 13804, XP055056523, DOI: 10.1073/pnas.1206906109 |
POSS, Z.CEBMEIER, C.C.ODELL, A.T.TANGPEERACHAIKUL, ALEE, T.PELISH, H.E.SHAIR, M.D.DOWELL, R.D.OLD, W.MTAATJES, D.J: "Identification of Mediator kinase substrates in human cells using cortistatin A and quantitative phosphoproteomics", CELL REP., vol. 15, 2016, pages 436 |
PUTZ, E.M.GOTTHARDT, D.HOERMANN, G.CSISZAR, A.WIRTH, S.BERGER, A.STRAKA, E.RIGLER, D.WALLNER, B.JAMIESON, A.M. ET AL.: "CDK8-mediated STAT1-S727 phosphorylation restrains NK cell cytotoxicity and tumor surveillance", CELL REP., vol. 4, 2013, pages 437 - 444 |
RAINEY, M.D.BENNETT, D.O'DEA, R.ZANCHETTA, M.EVOISIN, M.SEOIGHE, C.SANTOCANALE, C.: "ATR Restrains DNA Synthesis and Mitotic Catastrophe in Response to CDC7 Inhibition", CELL REP., vol. 32, 2020 |
RAINEY, M.D.QUACHTHITHU, H.GABORIAU, D.SANTOCANALE, C.: "DNA Replication Dynamics and Cellular Responses to ATP Competitive CDC7 Kinase Inhibitors", ACS CHEM. BIOL., vol. 12, 2017, pages 1893 - 1902 |
REMINGTON, J.P.JOSEPH P.OSOL, A., REMINGTON'S PHARMACEUTICAL SCIENCES, 1980, pages 1928 |
RZYMSKI, T.MIKULA, M.ZYLKIEWICZ, E.DREAS, A.WIKLIK, K.GOLAS, A.WOJCIK, K.MASIEJCZYK, M.WROBEL, ADOLATA, I. ET AL.: "SEL120-34A is a novel CDK8 inhibitor active in AML cells with high levels of serine phosphorylation of STAT1 and STAT5 transactivation domains", ONCOTARGET, vol. 8, 2017, pages 33779 - 33795, XP055817601, DOI: 10.18632/oncotarget.16810 |
SASI, N.K.BHUTKAR, A.LANNING, N.J.MACKEIGAN, J.PWEINREICH, M.: "DDK Promotes Tumor Chemoresistance and Survival via Multiple Pathways", NEOPLASIA, vol. 19, 2017, pages 439 |
SASI, N.K.TIWARI, K.SOON, F.F.BONTE, DWANG, T.MELCHER, K.XU, H.E.WEINREICH, M.: "The potent Cdc7-Dbf4 (DDK) kinase inhibitor XL413 has limited activity in many cancer cell lines and discovery of potential new DDK inhibitor scaffolds", PLOS ONE, vol. 9, 2014 |
SEO, J.O.HAN, S.I.LIM, S.C.: "Role of CDK8 and (3-catenin in colorectal adenocarcinoma", ONCOL. REP., vol. 24, 2010, pages 285 - 291 |
SHEU, Y.-J.STILLMAN, B: "Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression", MOL. CELL, vol. 24, 2006, pages 101 - 113 |
SHEU, Y.-J.STILLMAN, B: "The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4", NATURE, vol. 463, 2010, pages 113 - 117 |
STAHL, P.H.WERMUTH, C.G.: "Handbook of pharmaceutical salts : properties, selection, and use", vol. 446, 2011, article "International Union of Pure and Applied Chemistry" |
TEESDALE-SPITTLE, PJERRY MARCH: "Appl. Organomet. Chem.", vol. 7, 1993, WILEY, article "Advanced organic chemistry: Reactions, mechanisms and structure", pages: 293 - 293 |
TSHERNIAK, AVAZQUEZ, F.MONTGOMERY, P.GWEIR, B.A.KRYUKOV, GCOWLEY, G.S.GILL, S.HARRINGTON, W.F.PANTEL, S.KRILL-BURGER, J.M. ET AL.: "Defining a Cancer Dependency Map", CELL, vol. 170, 2017, pages 564 - 576 |
TSUTSUI, TUMEMURA, H.TANAKA, A.MIZUKI, F.HIROSE, Y.OHKUMA, Y: "Human mediator kinase subunit CDK11 plays a negative role in viral activator VP16-dependent transcriptional regulation", GENES TO CELLS, vol. 13, 2008, pages 817 - 826 |
VANOTTI, E.AMICI, R.BARGIOTTI, ABERTHELSEN, J.BOSOTTI, R.CIAVOLELLA, A.CIRLA, A.CRISTIANI, C.D'ALESSIO, RFORTE, B. ET AL.: "Cdc7 kinase inhibitors: pyrrolopyridinones as potential antitumor agents. 1. Synthesis and structure-activity relationships", J. MED. CHEM., vol. 51, 2008, pages 487 - 501, XP002584693, DOI: 10.1021/JM700956R |
WANG, B.WANG, M.ZHANG, W.XIAO, T.CHEN, C.-H.WU, A.WU, FTRAUGH, N.WANG, X.LI, Z. ET AL.: "Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute", NAT. PROTOC., vol. 14, 2019, pages 756 - 780, XP036714483, DOI: 10.1038/s41596-018-0113-7 |
WERMUTH, C.G.STAHL, P.H.: "IUPAC Pharmaceutical Salts: Properties, Selection, and Use A Handbook", CHEM. INT, vol. 24, 2002, pages 1 - 2 |
XI, M.CHEN, T.WU, C.GAO, X.WU, Y.LUO, X.DU, KYU, L.CAI, T.SHEN, R. ET AL.: "CDK8 as a therapeutic target for cancers and recent developments in discovery of CDK8 inhibitors.", EUR. J. MED. CHEM., vol. 164, 2019, pages 77 - 91, XP085584636, DOI: 10.1016/j.ejmech.2018.11.076 |
ZHANG, LCHENG, C.LI, J.WANG, L.CHUMANEVICH, A.A.PORTER, D.CMINDICH, A.GORBUNOVA, S.RONINSON, I.BCHEN, M. ET AL.: "A Selective and Orally Bioavailable Quinoline-6-Carbonitrile-Based Inhibitor of CDK8/19 Mediator Kinase with Tumor-Enriched Pharmacokinetics", J. MED. CHEM., vol. 65, 2022, pages 3420 - 3433 |
Also Published As
Publication number | Publication date |
---|---|
WO2023217980A1 (en) | 2023-11-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7377679B2 (en) | Combination therapy comprising a KRASG12C inhibitor and one or more additional pharmaceutically active agents for the treatment of cancer | |
Zhan et al. | MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma | |
AU2019374142A1 (en) | Methods of treating cancer in biomarker-identified patients with non-covalent inhibitors of cyclin-dependent kinase 7 (CDK7) | |
CA3177740A1 (en) | Kif18a inhibitors for treatment of neoplastic diseases | |
JP2019508404A (en) | Compounds, compositions and methods for stratification of cancer patients and cancer treatment | |
Cao et al. | Discovery of novel small molecule inhibitors of VEGF expression in tumor cells using a cell-based high throughput screening platform | |
US20210347761A1 (en) | Usp7 inhibition | |
US11267809B2 (en) | BAF complex modulating compounds and methods of using the same | |
Yan et al. | Design and synthesis of a 2-amino-pyridine derivative as a potent CDK8 inhibitor for anti-colorectal cancer therapy | |
EP4275686A1 (en) | Combination therapy for cancer comprising a cdc7 inhibitor and a cdk8 inihibitor or a ccnc inhibitor | |
WO2017190077A1 (en) | Ty-52156 compounds for the treatment of cancer | |
US20240238300A1 (en) | Treatment methods for subjects with cancer having an aberration in egfr and/or her2 | |
EP4183395A1 (en) | Pyrimidine compound-containing combination to be used in tumor treatment | |
US20240252454A1 (en) | Composition comprising an inhibitor of mitochondrial transcription | |
US20230338342A1 (en) | Methods of treating ribonucleotide reductase-related diseases with a ribonucleotide reductase inhibitor | |
US20230390251A1 (en) | Methods of treating ribonucleotide reductase-related diseases with a ribonucleotide reductase inhibitor | |
TW202202143A (en) | Co-treatment with cdk4/6 and cdk2 inhibitors to suppress tumor adaptation to cdk2 inhibitors | |
CA3216716A1 (en) | Composition comprising an inhibitor of mitochondrial transcription | |
WO2023063432A1 (en) | Treatment methods for subjects having cancer with a dysregulated mapk and/or pi3k pathway | |
WO2022082056A1 (en) | Dosing regimens for cyclin‑dependent kinase 7 (cdk7) inhibitors |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION HAS BEEN PUBLISHED |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |